Research Project On Gelatin

Chapter 3A Formulation and evaluation of absorbable gelatin sponges
Biodegradable haemostats [Absorbable gelatin sponges] and Hydrogels are a part of

modern wound management systems. Development of absorbable gelatin sponges, drug
loaded sponges, hydrogels and drug loaded hydrogels for advanced wound management
was investigated in this research project. The developed formulations were evaluated to
determine their potential for advanced wound management as described in the following
chapters.
Chapter 3A: Formulation and evaluation of absorbable gelatin sponges
Chapter 3B: Formulation and evaluation of drug loaded absorbable gelatin sponges
Chapter 4A: Formulation and evaluation of hydrogels
Chapter 4B: Formulation and evaluation of drug loaded hydrogels
3A Development of absorbable gelatin sponges as biodegradable haemostat

Absorbable gelatin sponges are used in various surgical procedures to assist cessation of
bleeding. It is currently believed that the haemostatic effect of the sponges is linked to
the sponge porosity and to their ability to absorb blood [Moony DJ, et.al; 1996].
Moreover, due to the porosity of the sponge, blood platelets are caught and the
coagulation cascade is activated transforming soluble fibrinogen into a net of insoluble
fibrin which stops the bleeding. Thus, the sponge structure is believed to be essential for
the mode of action of the sponge. Formulations of gelatin sponges are made by preparing
gelatin solution in water and beating it vigorously to form firm foam. This foam is then
dried and sterilized for using it as a surgical haemostatic sponge [Shu Tung Li; 1997].
Procedure for formulation of such therapeutic sponges is described in US patent of 1949.

This involved the preparation of sponges by air drying of the gelatin foam and by
controlling the temperature below 330C and 10 % relative humidity.
The method of preparation of gelatin sponges during this project work was selected
based upon some reported formulation and technology related factors such as [Silver FH;
1994].
1.The pore size requirement (60µm - 200µm)
It is suggested that pore size in the range of 60µm - 200µm is optimal for attachment to
the tissues and good fluid building capacity.
2.Uniformity and Wall thickness of pores
Design and development of surgical dressings for advanced wound management 91

Formulation and evaluation of absorbable gelatin sponges
Uniformly distributed pores with higher wall thickness give good fluid building capacity
when applied to bleeding sites.
3.Digestibility of sponge (< 75 min.)
This is an important criterion to check the digestion of sponge with proteolytic enzymes
secreted at the wound site. The digestibility of sponge is also an indication of
biocompatibility of sponge inside the body.
4.Water absorption (35x)
The sponges with high water absorption would also absorb more blood and other fluids
when applied to wound site.
5.Stability of product
The sponge should not absorb the moisture from atmosphere and its structure should
remain stable till its use.
6.Any toxic effects associated with final product
Since this product is absorbable inside the body and is biodegradable, it should not have
any toxic substances which can show adverse effects when applied to the wound site.
Gelatin, a natural protein is used in biodegradable materials. It is converted into other

forms such as sponges by crosslinking processes. Physical crosslinking of gelatin films is
carried out by thermal heating and ultraviolet irradiations. For chemical crosslinking of
gelatin several crosslinking agents such as formaldehyde, glutaraldehyde, water soluble
carbodiimides, diepoxy compounds, diisocynates are used [Tomihata, et.al; 2001]. These
agents form amide bridges within the amino acids present in the gelatin and cause
crosslinking of the gelatin. The toxicity of crosslinking agent should be taken into
consideration while development of gelatin sponges [Young SC, et.al; 1999].
The development of absorbable gelatin sponges in the present project was attempted by
designing many formulations using porcine grade of gelatin in concentrations ranging
from 1% to 5%. For crosslinking of the gelatin, less toxic crosslinking agents such as
formaldehyde, glutaraldehyde and 1- ethyl-3-(3-dimethylaminopropyl) carbodiimide
(EDC) were tried. The gelatin solution along with crosslinking agent was beaten
vigorously to form firm foam and this foam was dried to get the sponges [Silver FH;
1994].

Reaction of formaldehyde with gelatin

1. The aldehyde group can combine with nitrogen and some other atoms of gelatin or
with two such atoms if they are very close together, forming a cross-link -CH2- called
a methylene bridge. Studies of the chemistry of tanning process indicate that the most
frequent type of cross-link formed by formaldehyde in gelatin is between the
nitrogen atom at the end of the side-chain of lysine and the nitrogen atom of a
peptide linkage (Figure 3.1), and the number of such cross-links increases with time
[Gustavson; 1956]
2. The fixative action of formaldehyde is probably due entirely to its reactions with
proteins. Initial binding of formaldehyde to protein is largely completed in 24 hours
[Helander, 1994] but the formation of methylene bridges proceeds much more
slowly. Substances such as carbohydrates, lipids and nucleic acids are trapped in a
matrix of insolubilized and cross-linked protein molecules but are not chemically
changed by formaldehyde unless fixation is prolonged for several weeks [Ian D,
et.al; 1963].
Figure 3.1 Mechanism of crosslinking with formaldehyde
3A.1 Experimental
Gelatin (Porcine) was soaked in 100ml of water for 30 minutes to get solution at the
varying concentration of 1-6% of gelatin [Table3.1]. The suspension was stirred while
heating till gelatin was solubilised. The gelatin solution was then allowed to cool up to
o
40 C. Cross linking agent, formaldehyde varying in concentration of 0.1-2 mg/ml was

added. This solution was then stirred vigorously for 10 minutes by using overhead stirrer
to form firm foam. This foam was spread on trays and dried using the process of
lyophilization.
The method of preparation of gelatin sponges was optimized for various parameters such
as concentration of gelatin solution (1-6%w/v), stirring speed at low (500-1000rpm),
medium (1200-1400rpm) and high (1400-1500rpm) and concentration of cross linking
agent at the concentrations of 0.1, 0.3 and 0.5%w/v. Gelatin sponges obtained were
evaluated for physical characteristics. Compositions of formulation and effect of various
parameters are reported in table 3.2.
Gelatin foam was optimized and characterized for following parameters

3A.1.1 Characterization of gelatin foam
Optimum conditions for foam generation were determined by evaluating the resulting
foam for following parameters [Nagwa HF, et.al; 2007]:
Foam uniformity: by observing homogeneity of air bubbles under optical
microscope.
Foam stability: by observing separation of liquid part from derived foam in a
cylinder along 60 minutes after finishing foam generation.
Apparent foam density: by dividing foam weight (mg) over the foam volume (ml)
in the same cylinder.
Foam volume: reciprocal of apparent foam density.
Pictures depicting calculation of foam density and foam uniformity are shown in figure
3.2.
3A.1.2 Optimization of methods

Optimized method for production of gelatin foam should produce foam of pore size with
uniform size distribution with a mean pore size between 60-200μm having uniformity
from batch to batch. Additionally optimized formulation method should provide gelatin
sponge which will be physically and chemically stable as well as have high water
absorption capacity. All of the above mentioned objectives were met by optimizing
various formulation and process parameters.

Optimization of concentration of gelatin solution

For the purpose of optimizing the pore size and stability of the Gelatin foam, the process
was optimized for concentration and pH of gelatin solution. It is reported that, increase in
the pH of the gelatin solution causes increase in the viscosity of gelatin solution which
results in increase in cross linking of the gelatin at less concentration of cross linking
agent [Turner TD; 1979].Various concentrations of gelatin solutions 1, 2, 3, 4, 5 and 6%
(w/v) adjusted to pH values of 5.5, 6.5 and 7.5 using 0.1 N sodium hydroxide were
prepared for foam generation. Results are given in table 3.1.
Optimization of pore size and wall thickness

Pore size and wall thickness of gelatin sponges were optimized by optimizing
concentration of gelatin and formaldehyde as crosslinking agent.
Effect of concentration of gelatin and formaldehyde on pore size and wall thickness of
product was studied by plotting various combinations [Table 3.2] with surface response
curves and was interpreted in terms of % contribution of each factor from equations
obtained from Stat-Ease Design Expert v.7 software. Effect of gelatin concentration and
formaldehyde concentration on pore size and wall thickness are graphically shown in
figure 3.3 and their contribution affecting the gelatin sponge properties was calculated
from equations 1 and 2 obtained as a result of surface response methodology.
Optimization of whipping condition

Gelatin foams were produced by the whipping of solution. The mechanical stirrer (REMI
Motors) consisted of double blades with speed controller device that enhances air bubble
incorporation and foam stability. Solutions containing different concentrations of gelatin
were transferred to foam apparatus tank and generated into foams under varying
conditions. The effect of whipping duration (5, 10, 15 and 30 minutes) and speed of
stirring (low, medium and high) were investigated. Results are reported in tables 3.3 and
3.4.

a. Measurement
of foam density
and foam b. A photomicrograph, 200x, of 5%
volume. gelatin stable foam.
Figure 3.2 Measurement of foam density and surface characteristics
Table 3.1 Effect of concentration and pH of gelatin on stability and pore size and wall thickness of
gelatin foam
Formulation Gelatin solution

Code Concentration pH Viscosity Foam Pore size Wall thickness
(cps at stability (µm) (µm)
(% w/v)
100rpm)
GS1.1 5.5 20 Unstable - -
GS1.2 1 6.5 40 Unstable - -
GS1.3 7.5 100 Unstable - -
GS2.2 2 6.5 100 Unstable - -
GS2.3 7.5 260 Unstable - -
GS3.2 3 6.5 125 Unstable - -
GS3.3 7.5 300 Unstable - -
GS4.1 5.5 110 Stable 300-400 1-2
GS4.2 4 6.5 350 Stable 200-250 1-2
GS4.3 7.5 645 Stable 250-300 1-2
GS5.1 5.5 150 Stable 80-150 1.5-2.5
GS5.2 5 6.5 500 Stable 80-150 1.5-2.5
GS5.3 7.5 850 Stable 80-120 1.5-2.5
GS6.1 5.5 500 Unstable 80-150 2.5-3.5
GS6.2 6 6.5 1000 Unstable 80-150 2.5-3.5
GS6.3 7.5 1600 Unstable 80-120 2.5-3.5
Gelatin concentrations of 4% and 5% were found to give stable foam but 5%

concentration of gelatin resulting stable foam with small pore size of 80-150μm at the pH

of 5.5, 6.5 and 7.5; whereas gelatin concentration of 6% gave highly viscous solutions
with increase in pH which was difficult to formulate into foam.
Effect of gelatin concentration and formaldehyde concentration in the range of 0.1, 0.3
and 0.5%w/v on pore size and wall thickness is given in figure 3.3 as surface response
curves.
Figure 3.3 Surface response plots for optimization of a. Pore size and b. Wall thickness
Pore size (µm)= 29.25 + 56.90 x polymer conc. + 397.50 x formaldehyde conc. – 143.00 x polymer conc.
x formaldehyde conc ---------1
As seen in surface response curves, contribution of polymer concentration, crosslinking
agent and both the parameters on wall thickness were 85.90%, 8.77% and 0.036%
respectively; which was calculated from equation 1. Gelatin concentration is the

important parameter to be optimized which has maximum, 85.90% of effect on pore size
of the sponge.
Wall thickness (µm) = 0.25 + 2.25 x polymer conc. + 7.50 x formaldehyde conc. + 0.5 x polymer conc. x
formaldehyde conc ---------2
Contribution of polymer concentration, crosslinking agent concentration and both the
parameters on pore size were 0.022%, 3.10% and 49.56% respectively; which was
calculated from equation 2. For optimization of wall thickness of sponge, concentration
of gelatin and formaldehyde both should be optimized which together have the effect of
49.56% on wall thickness of sponge.
Formulation GS5.1, GS5.2 and GS5.3 having gelatin concentration of 5%, at pH 5.5, 6.5
and 7.5 was further optimized for concentration of formaldehyde in the range of 0.1, 0.3
and 0.5% w/v. The results for foam stability, pore size and wall thickness are given in
table 3.2.
Table 3.2 Effect of concentration of crosslinking agent on stability and pore size of gelatin foam
Formulation Gelatin solution Concentration of Wall thickness

code Concentration formaldehyde Foam Pore size (µm)
(% w/v) and pH (%w/v) stability (µm)
GS5.1 0.1 Unstable 80-150 2.5-3.5

GS5.1A 5 %, pH: 5.5 0.3 Unstable 80-150 3-5
GS5.1B 0.5 Stable 80-120 3-5
GS5.2 0.1 Unstable 80-150 2.5-3.5
GS5.2A 5 %, pH: 6.5 0.3 Unstable 80-150 3-5
GS5.2B 0.5 Stable 80-120 3-5
GS5.3 0.1 Unstable 80-150 2.5-3.5
GS5.3A 5 %, pH: 7.5 0.3 Stable 80-150 3-5
GS5.3B 0.5 Stable 80-120 3-5
Highlighted formulations in table 3.2 gave stable gelatin foams with pore size in the
range of 80-120 µm and wall thickness of 3-5 µm. Formulation GS5.3A having lowest
concentration of formaldehyde of 0.3%w/v was selected for further formulation of
gelatin sponge to avoid residual effects of formaldehyde.
Composition of formulation GS5.3A was further optimized for process parameters like
agitation speed of stirrer at different rpm, results are reported in table 3.3

Table 3.3 Effect of speed of agitation of stirrer on pore size of sponges
Speed of stirrer. Foam volume as

that of initial volume Observations
Mechanical stirrer. of gelatin solution
Low Pore size >300µm.
500-1000 rpm 2 times No uniform distribution of pores.
Medium Pore size >200µm.
1200-1400 rpm 3 times Homogenous distribution of pores.
For High Pore size within 100-200µm.
mula 1400- 1500 rpm 5 times Homogenous distribution of pores.
tion GS5.3A at high speed of 1400-1500rpm was found to give 5 times the foam of
original volume of gelatin solution with homogenous distribution of pores within size
range of 100-200μm. The formulations were further optimized for whipping duration by
checking gelatin foam properties at whipping duration of 5, 10, 15 and 30 minutes.
Results are reported in table 3.4.
Table 3.4 Effect of whipping duration on Gelatin foam properties

Formulation Whipping Apparent Foam Foam
Code duration density volume Foam uniformity stability
(min) (mg/ml) (ml/g)
GS5.3A 5 396 2.52 Nonhomogenous, large Unstable
air bubbles
GS5.3A 10 376 2.66 Nonhomogenous, large Unstable
air bubbles
GS5.3A 15 360 2.77 Homogenous, small air Stable
bubbles
GS5.3A 30 355 2.81 Homogenous, small air Stable
bubbles
Formulation GS5.3A after whipping at 1400-1500rpm for varying duration of time i.e. 5,
10, 15 and 30 minutes were found to give homogenous foam at whipping duration of 15
and 30 minutes. Whipping duration of 30 minutes was found to give apparent density of
355 and foam volume of 2.81 which was not significantly more than that of whipping
duration of 15 minutes; so formulation GS5.3A with whipping time duration of 15
minutes was selected for further preparation of gelatin sponges to reduce the
manufacturing time.
3A.1.3 Results and Discussion

Crosslinking of gelatin with glutaraldehyde is reported to give yellow and brittle sponges
and higher glutaraldehyde concentration was required for complete crosslinking. This
may cause irritation and toxic effects when applied to bleeding sites [MacLeod J; 1981].
EDC which is a biocompatible crosslinking agent also didn’t give sponges of desired

quality. The sponges obtained by using EDC as crosslinking agent were hard with poor
water absorption. To overcome such problems, other crosslinking agent, formaldehyde
was tried for the preparation of sponges [Cooes IH; 1969].
The properties and stability of gelatin foams are strongly affected by gelatin
concentration, its pH, concentration of crosslinking agent and conditions of
whipping process.
There was linear, gradual increase in gelatin solution viscosity upon increasing
gelatin concentration and the pH. Abrupt viscosity increase was observed after
5% of gelatin solution.
Stable homogenous gelatin foams with small (80-120 μm) size pores could only
be achieved from gelatin concentration of 5% w/v of gelatin solution at pH range
of 5.5 to 7.5 having wall thickness of 1.5-2.5μm. Drainage of liquid with foam
shrinkage and collapse of foam column were observed at lower gelatin
concentrations. Gelatin concentration higher than 5% were not suitable for foam
generation where the foams were very viscous and nonhomogenous (Table 3.1).
Lower or higher pH values lead to decreased foam rigidity and resulted in foam
lamella rupture.
Effect of gelatin and formaldehyde concentration on pore size and wall thickness
of sponges was determined by using surface response curve methodology (Figure
3.3). It was observed that gelatin concentration is the important parameter to be
optimized which has maximum, 85.90% of effect on pore size of the sponges and
wall thickness of sponge is dependent on concentration of gelatin and
formaldehyde both of which together have the effect of 49.56%.
Gelatin concentration of 5% at pH 6.5 and formaldehyde concentration of
0.5%w/v were found to give stable foam with pore size of 80-150μm whereas
same results were observed with 5% gelatin at pH 7.5 with 0.3% w/v of
formaldehyde concentration(Table 3.2). Lowest concentration of 0.3%w/v of
formaldehyde was selected for further formulation to avoid presence of any
residual formaldehyde which may cause toxic effects if remained after
crosslinking.
Lower stirring speed of 500-1000rpm was not able to produce stable foams.
Stirring at 1400-1500rpm with whipping duration of 30 minutes was required to
produce stable foam with lower foam density. Longer whipping for 30 minutes
did not significantly affect foam properties. Therefore, whipping 5% gelatin
solution at pH 7.5 for 15 minutes with stirring speed of 1400-1500rpm was found
to be optimum for stable gelatin foam generation.
Formulation combinations of GS5.3A was formulated into gelatin foam and freeze dried
as described in section 3A.2
3A.2 Drying of absorbable gelatin sponges by Lyophilization

(Freeze drying)
In the process of freeze drying, the drying occurs due to sublimation of ice. The solute in
the final product is relatively undisturbed during drying by lyophilization from that
originally in solution. The drying of gelatin foam was tried by lyophilization to avoid the
destruction of the porous structure of the foam and to get more uniform sponges with
desired pore size [Moony DJ; 1996].
Basic Understanding of Freeze-Drying Equipment and process

It is important to have a fundamental knowledge of the design and operation of the
freeze-drying equipment which is utilized for development and production of freeze-
dried products. Nail and Gatlin [1993] have provided an extensive discussion on the
design and operating components of most pharmaceutical freeze-dryers. These systems
consist of a chamber containing shelves through which a heat transfer fluid can be
circulated; a system for pumping, heating, and cooling the fluid; a vacuum pumping
system; a condenser for trapping water vapor; and a refrigeration system for cooling the
condenser. Additionally, most new freeze-dryers contain a system for sterilization of the
chamber and condenser.
Lyophilization takes place in three steps:
1. Prefreezing.
2. Primary drying.
3. Secondary drying.
The process was performed using LYOVAC Lyophilizer at laboratory scale [Figure 3.4].
Parts of Lyophilizer mentioned:
1.Condenser:
This keeps the temperature of the system to the set temperature.
2.Shelves:
Shelves are in the form of a circular plate in which frozen product is kept for drying.
3.Vacuum pump:
This part of the lyophilizer plays an important role in drying of the sponge.
Vacuum pump applies vacuum to the previously frozen foam kept in the shelves and
helps in drying by sublimation of ice from it to convert frozen foam in the form of
sponges.
Figure 3.4 Lyophilizer: LYOVAC [Laboratory Scale]
3A.2.1 Development and Optimization of lyophilization cycle

Although lyophilization process is widely accepted to manufacture stable drug products,
there are some challenges associated with each step of process. During freezing step,
significant difference in ice nucleation temperature exists within batch and also within
laboratory, pilot and manufacturing scale. Further, selection of an appropriate method to
determine end point of primary drying and determination of residual water is important
not only for process optimization but also for product quality assurance.
Lyophilization cycle:
The lyophilization, or freeze dry, cycle is the means by which a freeze dried product is
produced. A typical freeze dry cycle is described in terms of three phases - freezing,
primary drying and secondary drying. The freezing phase involves the immobilization of
water-ice and freeze-concentration of the product solution. The primary drying phase
involves removal of the water-ice by sublimation via application of high vacuum to the
chamber and input of heat to the lyophilizer shelves. Secondary drying describes removal
of unfrozen water from the product matrix or post-ice removal. Generally, the
lyophilization cycle is described in terms of the parameters which set the conditions
under which a product is lyophilized (e.g., shelf temperature and chamber pressure).
Alternatively, the freeze dry cycle can be thought of as the thermal profile which the
product undergoes during lyophilization to achieve a final dry product.
Lyophilization cycle for absorbable gelatin sponge was optimized by following steps.
1. Characterization of formulation to be freeze dried:
Determination of glass transition temperature (Tg):
Measurement of Glass Transition temperature is a method to characterize polymeric
materials. The glass transition is the temperature where the polymer goes from a
hard, glass like state to a rubber like state. DSC defines the glass transition as a
change in the heat capacity as the polymer matrix goes from the glass state to the
rubber state.
Glass transition temperature of gelatin was determined using DSC and was used as
the maximum allowable temperature for drying of gelatin formulation [Frank KB;
2004].
The DSC curve for porcine gelatin is shown in figure 3.5.
2. Design of freeze drying cycle [Frank KB; 2004]:
Freezing:
Temperature of freezing of the formulation was optimized by freezing the
formulation at various temperatures and observing difference in pore size of frozen
and dried product.
Pore size was measured using optical microscopy and SEM (Table 3.5).
Primary drying:
Primary drying was carried out below the maximum allowable temperature
determined using DSC. At the end of primary drying, the quantity of unfrozen water
in the formulation should be below 20%. End point of primary drying was
determined after measurement of moisture content of product by Karl Fischer
apparatus (Table 3.6).
Secondary drying:
At the end of primary drying, the quantity of unfrozen water in the formulation
should be below 1%. End point of secondary drying was determined after
determination of moisture content of product by Karl Fischer apparatus (Table 3.6).
Stable Gelatin foam was prepared as described in section 3A.1 and this foam was kept in
o o
a deep freezer for the temperatures ranging from - 20 C to - 40 C. Foam was further
o
dried in lyophilizer by keeping drying temperature 50 C (less than Tg) and by applying
vacuum. The pore size was measured with the help of Image Analyzer. Observations are
given in table 3.5.
Tg
o
55.60 C
Figure 3.5 DSC Thermogram of porcine gelatin
Table 3.5 Optimization of lyophilization cycle
Trial no. Lyophilization cycle Pore size of wet Pore size of dried
sponge (µm) sponge (µm)
o
Freezing temp: - 20 C
LT 1 o 150-200 > 200
Condenser temp: - 50 C
o
Heater temp: + 50 C
o
LT2 o 100-125 150 -200
o
Heater temp: + 50 C
Freezing temp: - 30oC
LT3 Condenser temp: - 50oC 80-100 100 - 120
Heater temp: + 50oC
o
LT4 o 80-110 90 -120
o
Heater temp: + 50 C
o
LT5 o 80-100 90-110
o
Heater temp: + 50 C
Trials LT4 and LT5 were found to give smaller pore sizes of wet sponge 80-110μm and
90-120μm of dry sponge. It indicates that there was no significant change in pore size
after drying of gelatin sponge at the conditions given in trial LT4 and LT5. Trial LT4
was used for further optimization due to freezing temperature of - 35oC which is 5oC
more that trial LT5 to save the electricity consumption.
End point for primary drying of prepared sponge was determined after the duration of 10
and 12hrs whereas for secondary drying, it was determined after 14 and 16 hrs of
Lyophilization at 2mbar vacuum. Results are given in table 3.6.
Table 3.6 Determination of end point of primary and secondary drying [Thickness of sponge: 1cm]
Duration of Moisture content of

Lyophilization (hrs) gelatin sponge (%)
10 30
12 18 Primary drying complete
14 8
16 0.5 Secondary drying complete
Vacuum: 2 mbar. Drying time: depending upon thickness of sponge
3A.2.2 Results and discussion

 Lyophilization cycle for drying of gelatin sponges was developed by optimizing
time and temperature of freezing, primary drying and secondary drying.
o o
 Freezing temperatures of - 35 C and - 40 C were found to give pore size in the
range of 80 -100 µm, which was maintained even after drying of sponge (Table
o
3.5). Temperature of - 35 C was selected for the freezing to reduce the electricity
wastage since there was no significant difference in the properties of the sponge
o o
at - 35 C and - 40 C.
 Primary drying temperature was decided by determining glass transition
temperature (Tg) with the help of DSC studies to prevent collapse of porous
o
structure of formulation. It was observed that Tg of porcine gelatin was 55.60 C
(Figure 3.5), so the primary drying temperature for gelatin sponge was kept at 50
o
C which was below Tg of gelatin.
 Residual moisture content in lyophilized product was found to be 18% indicating
completion of primary drying after 12 hrs of lyophilization while it was found to
be 0.5% after 16hrs of lyophilization indicating complete drying of gelatin foam
having 1mm thickness. The drying time for sponge varied with the thickness of
sponge due to increase or decrease in water content with thickness of sponge.
After optimization of all the parameters for development of gelatin foam and drying of
gelatin foam; formulation of gelatin sponges was carried out by selecting the formulation
GS5.1A and the procedure mentioned in experimental section 3A.1. Drying of sponge
was carried out by the process of lyophilization utilizing the optimized trial as mentioned
in section 3A.2.
3A.3 Characterization and evaluation of plain gelatin sponges

Initial characterization of gelatin sponge is an important parameter which helps to select
suitable methods and excipients for development of final product. The sponges have
microporous structure which depends on the method adopted for the preparation and
drying of sponges. Gelatin sponges fabricated were characterized using following
techniques for preliminary studies and for the comparison of the products made under
different processing conditions.
3A.3.1 In-vitro characterization

The absorbable gelatin sponges were characterized according to the monographs in
United States Pharmacopoeia (USP) and British Pharmacopoeia (BP) standards. There is
some difference in procedures given in the two pharmacopoeias.
The following tests were carried out for the characterization of Gelatin sponge (Batch
GS5.1A) as per USP and BP monograph. The results of in vitro characterization are
given in table 3.9.
Appearance and physical characteristics

The appearance and physical characteristics of the gelatin sponges were checked by
visual observation and microscopy. Cross-section of gelatin sponge was taken and
mounted on the glass slide with a coverslip and observed using optical microscope under
10x and 65x lens. Photomicrograph of batch GS5.1A is shown in figure 3.7a.
Loss on drying
It was calculated to check the moisture content of the Gelatin sponge. The moisture
o
content was determined after drying at 105 C for 16 hours. Results are given in table
3.9.
Water absorption
Absorbable gelatin sponge should have good water absorption capacity so that it can
hold more quantity of blood when applied on the bleeding site. The absorbability of the
sponge depends on its cross linking degree and pore size. This test was performed based
on the USP procedure as described below. Results are given in table 3.9.
Procedure
A portion of about 1x1cm of sponge was cut, weighed accurately and placed in a beaker
containing 50ml of water. It was kneaded gently between the fingers until thoroughly
wet and until all the air has been removed taking care not to break the tissue. The portion
of the sponge was lifted from the water and blotted twice firmly between two pieces of
absorbent paper. The expressed sponge was dropped into a tared weighing bottle
containing about 20ml of water with a suitable hooked instrument, again took back after
water absorption and allowed to drain over the weighing bottle for 5 seconds. Again the
bottle containing water was weighed. The loss in weight of water represents the weight
of water absorbed by the sponge.
Morphology of sponges
In general, pore size, mean pore size and orientation of porous matrix are indispensable
elements of biologic activity of biomaterials having open-pored structures. The effects of
freezing rate on the sponge morphology are reported in many works [Thomas WP, et.al;
2002].
o
Quenching at -35 C could produce a lot of small crystals, resulting in the relatively
homogeneous morphology. So the sponges obtained by process of lyophilization give
sponges of homogeneous morphology [Thomas WP, et.al; 2000].
Morphology of gelatin sponge mainly depends upon concentration of gelatin,
crosslinking agent, freezing rate and drying method for sponge.
Procedure:
Morphology was observed with the help of Image Analyzer by mounting the cross-
section of sponge on the slide. It was also seen with the help of Scanning Electron
Microscopy.
The images were examined for the pore size and distribution of pores along the matrix.
The morphology of the prepared gelatin sponge was compared with marketed sponges,
AbGel from Mumbai and Surgifoam from Germany.
The images of gelatin sponge obtained from Image Analyzer are shown in figure 3.7a.
SEM images are shown in figure 3.7b, 3.7c and 3.7d respectively for gelatin sponge,
AbGel and Surgifoam.
i. Pore size
Pore size of the sponge is one of the main criteria for characterization of sponges. More
uniform the pore size, the sponges will show better absorbability. Pore size of the sponge
was determined using optical microscopy. The image of gelatin sponge by optical
microscopy is shown in figure 3.7a.
ii. Wall thickness

Wall thickness plays important role in fluid building capacity of sponge. More the
thickness of sponge more will be the fluid building capacity and there are less chances of
breaking up of structure of sponge.
Wall thickness of sponges was measured with the help of Image Analyzer. The images
obtained from Image Analyzer are shown in figure 3.7a.
Residual formaldehyde content

Formaldehyde is irritant to the eyes, nose and respiratory tract and may cause coughing,
bronchitis and pneumonia. Concentrated solution of formaldehyde when applied on the
skin can cause whitening and hardening of the skin. It can often cause contact dermatitis
and sensitivity reactions.
Formaldehyde was used as cross linking agent for the preparation of gelatin sponge. To
avoid all these adverse effects of formaldehyde the residual content of formaldehyde in
the sponges was checked by using chromotropic acid solution. The sponges prepared
should pass the test for residual formaldehyde content described in BP 93 to avoid any
adverse effects.
Procedure:
Piece of sponge weighing about 50mg was added to 100ml of water and macerated for at
least 2 hours, shaking occasionally. About 0.5ml of the supernatant liquid was
transferred to a glass stoppered test tube and 10 ml of chromotropic acid solution was
added, the tube was stoppered and heated in a water bath for 30 minutes.
The absorbance of the resulting solution was taken at 570nm. It should not be more than
that of a solution obtained by repeating the operation using 0.5 ml of solution containing
0.001% w/v of formaldehyde solution in place of supernatant liquid. Results are given in
table 3.9.
Digestibility
Absorbable gelatin sponge is a biodegradable material which is used as haemostatic in
surgical injuries. It should get digested inside the body without leaving any metabolic
product which may be harmful to the body. The sponge when applied to the wound site
gets absorbed inside the body and digested with the help of proteolytic enzymes at the
site.
The digestibility of the sponge was checked using pepsin which is proteolytic in 0.1N
Hydrochloric acid. The test of digestibility was carried out as per the method described
in BP 93.
Procedure:
A piece of prepared gelatin sponge weighing about 50mg was placed in a beaker
containing water. The piece was kneaded gently between the fingers until thoroughly wet
and until all the air has been removed, taking care not to break the tissue. Sponge piece
was lifted from the water and excess water was removed with absorbent paper. The
wetted sample was placed in a 150ml flask that contained 100 ml of 1% solution of
pepsin in 0.1N HCl and agitated gently and continuously until digestion was complete.
The average digestion time of three determinations was taken as the reading. Results are
given in table 3.9.
FT-IR spectroscopy
This study was carried out to determine crosslinking of the gelatin sponge by detecting
presence of amide linkage.
FT-IR spectra between 4000 cm-1 and 400 cm-1 of gelatin and crosslinked gelatin
sponges were obtained using KBr disc by using FTIR spectrophotometer [Jasco]. The
smoothing of spectra and the base line correlation procedure was used. Results are
shown in figure 3.8A and 3.8B.
Measurement of degree of Crosslinking

The degree of crosslinking was measured by modifying the method of Bubnis et.al
[1992]. TNBS labeled ε-amino groups present in gelatin were detected as per the method
described below [Habeeb AF, et.al; 1966, Snyder SL, et.al;1975].
Procedure
Approximately 11 mg of sponge sample was placed in a 50 ml screw cap test tube. One
milliliter of 4% NaHCO3 (pH 8.8) and 1.00 ml of 0.50% TNBS were added to the tubes.
o
The reaction mixture was heated at 40 C for 4 h with mild shaking. Three milliliter of 6
o
N HCl was added and mixture was autoclaved at 120 C and 15-17 psi for 1 h to
hydrolyze and dissolve any insoluble matter. The hydrolysate was then diluted to 5.0 ml
with water. The hydrolysate dilution was extracted with three 20 ml portions of ethyl
ether to remove excess unreacted TNBS. A 5.0 ml aliquot of the aqueous phase was
removed and heated for 15 min in hot water bath to evaporate residual ether. The aliquot
was diluted with 15.0 ml of water and the absorbance was measured at 346 nm using
Jasco double beam spectrophotometer.
The crosslinking degrees are obtained from the differences between the absorbance
values before and after crosslinking [Kale RN, et al; 2010].
The equation is as follows:
Absorbance of crosslinked sponge

Crosslinking degree (%) = 1- x 100
Absorbance of non-crosslinked sponge
After determination of degree of crosslinking, residual formaldehyde content for each

formulation was determined and safe and effective concentration of crosslinking agent
was determined for development of crosslinked gelatin sponge. Results are represented
graphically in figure 3.9.
In-vitro biodegradation of gelatin sponges

Biocompatibility of the material and their degraded products is a prerequisite for
resorbable haemostatics. In-vitro biodegradation studies for gelatin sponges are carried
out to check the in-vivo biocompatibility of the material. Gelatin is the denatured
collagen and primary structure of collagen molecule is homologous to human collagen.
Because of this property, gelatin sponges are used as biodegradable haemostatic
materials [Anderson JM, et. al; 1984].
It is known that a number of cell types such as polymorphonuclear leukocytes,
fibroblasts and macrophages during the wound healing period are capable of secreting
enzyme collagenase which cleaves collagen molecule at ¼th position from the C-
terminal end of the molecule [Suzuki Y, et.al; 1999].
The enzyme first reduces collagen molecule to two smaller triple helices which are not
stable at body temperature and are subsequently denatured to random coiled
polypeptides. These polypeptides are further degraded by proteases into amino acids and
short peptides that are metabolized through normal metabolic pathways [Suzuki Y, et.al;
1999].
Since gelatin is the denatured collagen, pepsin breaks down the amino acid chain of the
gelatin to smaller peptides and then to amino acids and short peptides which are
metabolized through normal metabolic pathway. So pepsin which is a proteolytic
enzyme is used to study the in-vitro biodegradation behavior of gelatin sponges
[Anderson JM, et.al; 1984]. General degradation mechanism for gelatin sponge is given
in figure 3.6.
Gelatin (sequence of amino acids)
In injury:
Polymorphonuclear leukocytes,
Leukocytes, fibroblasts &
Macrophages.
Breakdown
Secrete proteolytic enzymes.
Random coiled polypeptides

(Not stable at body temperature)
Amino acids & Short peptides

(Metabolized through normal metabolic pathway)
Figure 3.6 General degradation mechanism for gelatin [Shu TL; 1997]
Accordingly, experiment was designed to check the in vitro biodegradation of gelatin

sponge by using pepsin as a proteolytic enzyme causing degradation of gelatin as it
would cause in human body. The degradation behavior of Gelatin sponges were checked
using phosphate buffered saline (PBS) and pepsin solution and compared with marketed
Gelatin sponges.
Experimental:
Pieces of sponges (1cm x 1cm x 0.3cm) were immersed in 5ml of phosphate buffered
saline (PBS pH 7.4) containing 6 µg ml-1 of pepsin [Correll JT, et.al;1945]. After
o
incubation at 37 C, the sponges were repeatedly washed with distilled water and freeze
dried. This was carried out after every 6, 12, 24, 36, 60 and 72 hour. The changes in
weight of sponges were monitored after each time interval. This test was carried out on
developed gelatin sponge and both marketed (Local and Germany) gelatin sponges. The
graphs of degradation time (hours) versus weight remaining (%) were plotted and results
were compared. Comparative biodegradation behaviour of prepared gelatin sponge,
AbGel and Surgifoam is shown in figure 3.10.
Sterilization of gelatin sponges

Gelatin sponges are used as haemostatic agents. When applied as a haemostatic, the
sponge is placed on or in the body, and accordingly, the composition must be sterile at
the time of use.
Sterilization is an important consideration in the design of surgical dressings which are

used as bioabsorbable materials. Aseptic processing and terminal sterilization are the two
major routes for preparing sterile products. Aseptic processing involves the use of sterile
solvents in the clean room environment under GMP conditions [Avis KE, et.al; 1987].
Terminal sterilization involves [Dosimetry; 1973]:
 Dry heat sterilization or
 Gamma radiation
Conventionally, sterilization of such sponges is carried out at elevated temperatures for
o o
prolonged periods of time, e.g. 130 C to 140 C degree for 3 hours as described by
Correll [Correll JT, et.al; 1985]. The resulting heat sterilized product exhibites higher
tensile strength and significantly less fluid absorption (e.g. water, blood etc.) as
compared to the pre-sterilized product [Mekane, et.al; US Patent 2001].
It has been reported that packaged gelatin sponges can be sterilized by using gamma
radiation technique without affecting absorbability at the dose of 2.4 Mrad. Hence
gamma radiation was attempted for sterilization of developed sponges.
Procedure
After characterization and evaluation, sponges of size 10 x 10 x 7 cm were packed
individually in polythene first and then packed with the help of aluminium foil and
subjected to sterilization with γ irradiation. The packing selected will protect the sponge
from moisture. Since it is a lyophilized product, it is more prone to absorb the moisture.
The fine packing of sponge for sterilization is shown as following pictures.
Prepared Gelatin Sponge Packing of prepared gelatin sponge

Packed sponges were sterilized by 2.4 Mrad dose of radiation using γ-rays from 60Co at
ISOMED, BARC, Mumbai.
The formulations before and after sterilization were compared for following parameters:
1. Appearance
2. Water absorption
3. Pore size
4. Digestibility
5. In-vitro biodegradation
The tests were performed as per the procedures given in section 3A.3.1. Morphology of
sponges after sterilization was studied with the help of Image Analyzer. Comparative
images are given in figure 3.11.
The results of evaluation of sponges before and after sterilization are listed in table 3.8.
Comparative in vitro biodegradation before and after gamma sterilization are mentioned
in figure 3.12.
Sterility testing
After sterilization, 1x1cm pieces of selected formulations were subjected to sterility
testing as per IP procedure [IP; 2007].
Procedure
Using sterile forceps, 1x1cm of sponge from sterilized formulations was placed in fluid
thioglycollate medium and soyabean casein digest medium and incubated for 14 days.
The tubes were observed for any microbial growth at regular time intervals of 1,3,5,7 and
14 days. The tests were performed in duplicate. The results are shown in table 3.7.
Comparison with marketed gelatin sponges

After characterization and evaluation of developed gelatin sponge, the properties of the
sponge were compared with available marketed sponges such as AbGel from Mumbai
and Surgifoam from Germany. The results of comparative evaluation are given in table
3.10 and comparative in vitro biodegradation is shown in figure 3.13.
3A.3.2 Results and discussion

Morphology of sponges
Morphology of prepared and marketed gelatin sponges is shown in figure 3.7. The pore
size distribution and wall thickness of developed gelatin sponge was found to be within
60-120 μm and 3-5 μm respectively. These are quite similar to marketed gelatin sponge,
Surgifoam. AbGel was found to show pore size in the range of 100-120 μm which was
larger than developed gelatin sponge and Surgifoam.
A Developed gelatin sponge

a. Photomicrograph (100x) b. SEM image (250x)
c. SEM (100x): AbGel gelatin sponge d. SEM (300x): Surgifoam gelatin ponge
Figure 3.7 Comparative morphology of gelatin sponges
FT-IR spectroscopy for determination of crosslinking
Figure 3.8 FT-IR spectra of gelatin and crosslinked gelatin sponge
A. FTIR spectra of crosslinked uncrosslinked gelatin where peak assignment is as follow:

3500: Primary & secondary amines, 2885: C----H, 1112: CH2 and other groups
B. FTIR spectra of crosslinked gelatin sponge where peak assignment is as follow:

3381-3502: Primary & secondary amines, 3142: Amines, 2926-2955: Aldehydes / Alkanes
2332: N-H stretching, 1539-1637: Amides (N-H)/C-N linkage / ester linkage
FT-IR spectroscopic analysis of uncrosslinked gelatin and crosslinked gelatin sponges

are shown in figure 3.8A and 3.8B. The representative absorption peaks in all samples
are assigned to amide I (1637cm-1), amide II (1539cm-1), amide III (1491cm-1) and ester
bond (1105cm-1).We noted the slight increase of amide I, amide II, and amide III, and
presence of peptide linkage formed due to reaction of aldehydes and amines. Presence of
peak at 2926- 2955cm-1 indicates free aldehyde groups which are not utilized for
crosslinking.
Measurement of degree of Crosslinking

The crosslinking degree measured by TNBS assay showed an overall increasing
tendency to increase with amount of formaldehyde in the reaction medium.
Formaldehyde concentration of 0.05%w/w was found to show higher crosslinking degree

and 20% crosslinking degree at formaldehyde concentration of 0.03%w/w was found to
give crosslinked sponges having residual formaldehyde content within specified limits
(BP 1993) with higher water absorption capacity of 45g/g of sponge. Results are shown
graphically in figure 3.9.
50
40
30
20
10 Crosslinking degree (%)

Water Absorption (g/g)
0
0 0.02 0.04 0.06
Formaldehyde Conc. (wt%)
Crosslinking degree and water absorption was measured

as described in section 3.3
Figure 3.9 Crosslinking degree and water absorption Vs formaldehyde concentrations
In-vitro biodegradation of gelatin sponges

Developed gelatin sponges were further checked for in vitro biocompatibility by
evaluating in vitro biodegradation in pepsin solution. Total degradation of prepared
gelatin sponge was found to be within 72hrs as shown in figure 3.10.
In vitro biodegradation of Gelatin sponge

(Prepared)
100
80
Weight remaining(%)
60
40
20
0
0 12 24 36 48 60 72
Degradation time (hours)
Figure 3.10 In-vitro biodegradation of Prepared Gelatin Sponge

It was observed that developed plain gelatin sponge gets degraded totally in 72 hours.
The degradation was faster after 24 hours. Only small mass of gelatin remained after 60
hours which also degraded totally within 72 hours.
Sterilization of gelatin sponges

The sponges were sterilized by gamma radiation sterilization at the dose of 2.4Mrad. The
sponges were characterized after Gamma radiation sterilization to determine if any
effects of radiations. It did not show any significant changes in its characteristics such as
water absorption, pore size and digestion time. Sterilization by gamma radiation
technique was found to maintain the characteristics of the sponges so this method can be
employed for sterilization of surgical sponges. Results of comparative characterization of
gelatin sponge before and after sterilization are given in table 3.7, 3.8 and figure 3.11,
3.12.
Table 3.7 Test for sterility performed on sterilized gelatin sponges
Sr.no. Medium Observation for 14 days.

1. Fluid Thioglycollate Medium No microbial growth seen.
2. Soyabean Casein Digest Medium No microbial growth seen.
Table 3.8 Characterization of Gelatin sponge before and after gamma sterilization
Sr.no. Tests Before sterilization After sterilization

1. Appearance White, Porous White, Porous
2. Water absorption 45-50 times 45-50 times
3. Pore size 80-110µm 80-110µm
4. Digestibility <10 minutes <10 minutes
5. In-vitro biodegradation <72 hours <72 hours.
Before sterilization After sterilization

Figure 3.11 Images from Image Analyzer
Porous structure of sponge when observed using image analyzer, did not show
significant difference in images before and after sterilization.
In vitro biodegradation behavior of sterile sponge did not show any significant difference
from unsterile sponge. Both the sterile and non-sterile sponges degraded totally within 72
hours as shown in figure 3.12.
100
Weight remaining (%)

80
60
40
20
0
0 12 24 36 48 60 72
Degradation time (hrs)
Weight remaining (%) before sterilisation
Weight remaining (%) after serilisation
Figure 3.12 Comparison of in vitro biodegradation of sponge before and after sterilization
Table 3.9 Characterization and evaluation of prepared plain gelatin sponge
Parameter Observations
Appearance White, porous,
sponge like.
Solubility Insoluble but
swells in water.
Sterility Sterile.
Loss on drying 10%
Digestibility 50 min.
Water absorption 45-50
Formaldehyde < 0.001%
Pore size 80-120um
Wall thickness 3-5um
Comparison with marketed gelatin sponges

Optimized batch of developed gelatin sponges was compared with existing formulations
of gelatin sponge, Abgel; available in Indian market and Surgifoam; available in
international market. Comparative characteristics are given in table 3.10.
Table 3.10 Comparison of characteristics of prepared gelatin sponge with marketed formulations,
AbGel and Surgifoam
Parameter Prepared sponge Local market German market

(GS5.1A) (AbGel) (Surgifoam)
Appearance White, porous, White, porous, White, porous,
sponge like. sponge like. sponge like.
Solubility Insoluble but Insoluble but Insoluble but
swells in water. swells in water. swells in water
Sterility Sterile. Sterile. Sterile.
Loss on drying 10% 12% 8%
Digestibility <10 min. <10 min. <10 min.
Water 45-50 times 35-40 times 45-50 times
absorption
Residual < 0.001% < 0.001% < 0.001%
formaldehyde
Pore size 60-120 um 100-120 um 80-110 um
Wall thickness 3-5 um 2-4 um 3-6 um
In vitro biodegradation of Gelatin sponge

(Comparison)
100
80
60
40
20
0
0 12 24 36 48 60 72
Degradation time (hours)
Weight remaining (% ) Local Weight remaining (% ) Germany.
Weight remaining(% ) Prepared
Figure 3.13 Comparison of in vitro biodegradation of Gelatin Sponges
Developed gelatin sponges found to exhibit comparable properties with the AbGel and
Surgifoam available in Indian and International market respectively.
The characteristics of developed sponges (Table 3.8) were found to be
comparable with marketed AbGel and Surgifoam sponge. All the three sponges
were digested within 10 minutes. Developed gelatin sponge absorbed 45-50 times
of water to its initial weight. AbGel was less absorbable i.e. 35- 40 times and
water absorption of Surgifoam was 45-50 times. Both developed gelatin sponge
and Surgifoam had similar water absorption capacity.
Degradation of AbGel also occured within 72 hours. But degradation was faster
than that of developed gelatin sponges. Weight loss was more than that of
prepared gelatin sponge. Only very small fibrous mass remained after 60 hours
which totally degraded within 72 hours. It may be because of less crosslinking of
gelatin.
Biodegradation of Surgifoam occured similarly as that of developed gelatin
sponge. It totally degraded in 72 hours showing comparable weight loss with
developed sponges. Surgifoam and developed gelatin sponges were found to
show comparatively similar biodegradation behaviour. Prepared gelatin sponges
were found to biodegrade totally in 72 hours and have comparable properties
with marketed sponges.
After in vitro characterization, Optimized formula of gelatin sponge GS5.3A, was

taken up for scale up studies. Scale up studies is described in section 3A.4.
3A.4 Scale up and reproducibility studies

Introduction:
Scale up or pilot plant operations are performed to produce larger quantities of products
than are reasonably available from laboratory scale facilities and to predict engineered
manufacturing costs. Larger quantities of sponges were required to initiate [Michael
Levin; 2005]:
Product performance tests.
Manufacturing assessments.
Manufacturing equipment performance validation.
Scale up is generally defined as the process of increasing batch size and a procedure for
applying the same process to different output volumes.
For the gelatin sponge formulations, the scale up and reproducibility studies were
performed on the optimized formula of gelatin sponge and the process was validated.
Scale up studies
Scale up studies for absorbable gelatin sponge were carried out at two stages
1. Stage 1: Pilot scale.
2. Stage 2: Commercial scale.
To achieve the scale up at both the stages, study was divided into two major steps of
I. Formulation process scale up and
II. Lyophilization process scale up
3A.4.1 Formulation process scale up

Success for scale-up is when geometrically similar process and processing equipment are
used at each manufacturing scale. Geometric similarity means that equipment shape and
dimensions are proportional from one production scale to another. Geometric similarity
would ensure results that are independent of scale. Equipment manufacturers often tout
the scalability of their equipment, specifically referring to the geometric similarity of
various equipment sizes [Michael Levin; 2005].
For scale up of formulation process, initially all the variables that were thought to be
crucial for the process were listed out for optimization [Lawrence HB; 2005]. These
variables were then optimized by evaluation and determination of desired end point as
given in table 3.11. Stirrer and manufacturing vessel used for making the formulation
were scaled up in geometric proportion as given in table 3.12.
Optimum conditions for foam generation were determined by evaluating the resulting
foam for foam uniformity, foam stability, apparent foam density and foam volume as
mentioned in section 3A.1.1.
As described in section 3A.1, concentration of gelatin, crosslinking agent and whipping
conditions for preparation of gelatin sponge are the main variable factors for
development of formulation of gelatin sponges.
Table 3.11 Variables for Process scale up of Absorbable gelatin sponge:
Variables End point determination Desired end point

Gelatin concentration Foam stability Stable
Crosslinking agent conc. Foam density <400 mg/ml
Whipping conditions Foam volume 4-5 ml/mg
3A.4.2 Design of manufacturing vessel

Depending on the results obtained for formulation development of gelatin sponges,
manufacturing vessel was designed for two scale up stages from laboratory scale.
1. Stage 1: Pilot scale. (Basis: 20 pieces of 8 x5 x1cm size; Gelatin solution:
250ml)
2. Stage 2: Commercial scale. (Basis: 60 pieces of 8 x5 x1cm size; Gelatin
solution: 500ml)
Manufacturing vessel for scale up of gelatin sponge formulation was designed by taking
dimensions of laboratory scale manufacturing vessel into consideration. Scale up
manufacturing vessel should have dimensions in proportion with original dimensions of
laboratory manufacturing vessel to maintain geometrical similarity [Michael Levin;
2005].
Scale up of gelatin sponge manufacturing process was carried out at two stages to
prepare 20 pieces and 60 pieces of 5cm x 8cm x 1cm of gelatin sponge from the
laboratory stage of 5 pieces of 5cm x 8cm x 1cm of batch of gelatin sponge.
The dimensions of manufacturing vessel and stirrer used at two stages of manufacture
were varied to maintain geometrical similarity as mentioned in table 3.12.
Table 3.12 Dimensions of manufacturing vessel and stirrer used for scale up
Manufacturing vessel Measurement (inch)

(SS tank)
Dimensions Lab scale Scale up stage 1 Scale up stage 2
Diameter 10cm 12cm 16cm
Height 13cm 23cm 43cm
Material of construction S.S. 316 S.S. 316 S.S. 316
Mechanical stirrer Measurement (inch)
Dimensions Lab scale Scale up stage 1 Scale up stage 2
Shape of blade Round Round Round
Diameter of blade 4cm 6cm 10cm
Number of blades 1 2 2
Optimization of whipping conditions for scale up

Along with design of manufacturing vessel, scale up of gelatin sponge was optimized for
whipping conditions as stage 1 and stage 2. Stable Gelatin foam was prepared as
described in section 3A.1 by making 250ml gelatin solution for scale up at stage 1 and
500ml gelatin solution for scale up at stage 2. For optimizing whipping conditions,
concentration of gelatin was varied at 5 and 6% and duration of whipping was varied at
5, 10, 15 and 30 minutes. Other parameters such as concentration of formaldehyde
(0.3%w/w) and agitation speed (1400-1500rpm) were kept same those used for
formulation GS5.3A (table 3.2). Observations of foam stability at two scale up stages are
reported in table 3.13.
Table 3.13 Optimization of whipping conditions for scale up
Formul Gelatin Whipping Apparent Foam Foam

-ation conc. duration density volume Foam uniformity stability
(% (mg/ml) (ml/g)
Code (min)
w/v)
Stage 1: Basis: 20 pieces of 8 x5 x1cm size
GSC1 5 400 2.27 Nonhomogenous, large air bubbles Unstable
GSC1.1 5 10 374 2.67 Nonhomogenous, large air bubbles Unstable
GSC1.2 15 361 2.77 Homogenous, small air bubbles Stable
Stage 2: Basis: 60 pieces of 8 x5 x1cm size
GSC3.2 15 378 2.65 Nonhomogenous, large air bubbles Unstable
GSC4.2 15 415 2.41 Nonhomogenous, large air bubbles Unstable
Formulation GSC1.2 and 1.3 after whipping at 1400-1500rpm at varying duration of 5,

10, 15 and 30 minutes were found to give homogenous foam at whipping duration of 15
and 30 minutes. Whipping duration of 30 minutes was found to give apparent density of
358 and foam volume of 2.79 with stable foam having homogenous air bubbles, which
was not significantly more than that of whipping duration of 15 minutes; as well as
gelatin concentration of 6%; formulation GSC2.2 and 2.3 were similar to laboratory
scale formulation. Formulation GSC1.2 with duration of 15 minutes was selected for
preparation of gelatin sponge to reduce the manufacturing time at scale up stage 1 to
produce 20pieces of 8x5x1cm of gelatin sponge per cycle.
For scale up stage 2, Formulation GSC3.3 after whipping at 1400-1500rpm at varying

duration of 5, 10, 15 and 30 minutes was found to give stable homogenous foam at
whipping duration of 30 minutes with apparent density of 360 and foam volume of 2.78.
Observations for formulation GSC3.4 at 6% of gelatin were not significantly different
than that GSC3.3. The foam properties of formulation GSC3.3 and GSC1.2 of scale up
stage 1 were similar. Formulation GSC3.3 with duration of 30 minutes was selected for
preparation of gelatin sponge at scale up stage 2 to produce 60 pieces of 8x5x1cm of
gelatin sponge per cycle.
3A.4.3 Lyophilization process scale up

The goal during lyophilization scale-up is to maintain equivalent product temperatures or
the same product "thermal history" between laboratory and commercial processes. This
philosophy is rooted in the belief that a similar product temperature profile should
provide the same product characteristics and, subsequently, the same stability profile
[Serguei T; 2007].
Successful scale-up of a lyophilization cycle from laboratory to pilot or commercial scale
requires an understanding of relative performance characteristics of the lyophilizer at
each scale. These characteristics may significantly alter the product temperature profile
upon scale-up if not properly considered.
The Lyophilization, or freeze dry, cycle is the means by which a freeze dried product is
produced. A typical freeze dry cycle is optimized at all the three phases - freezing,
primary drying and secondary drying [Stanley M, et.al; 2007].
The freezing phase involves the immobilization of water-ice and freeze-concentration of
the product solution. The freezing stage of lyophilization provides the foundation for a
lyophilized product. During freezing, the cake pore structure is established. The
resistance to water flow (mass transfer) from the pore structure is a key product property
from a cycle characterization point of view [Stanley M, et.al; 2007].
The primary drying phase involves removal of the water-ice by sublimation via
application of high vacuum to the chamber and input of heat to the lyophilizer shelves. It
maintains the porous structure of product. Collapse of the porous structure of the product
is prevented by optimizing the proper temperature for primary drying [Stanley M, et.al;
2007].
Secondary drying describes removal of unfrozen water from the product matrix post-ice
removal. Optimization of this step is needed to assure complete removal of moisture
content of the product which can affect its stability [Stanley M, et.al; 2007].
3A.4.3.1 Lyophilization cycle scale up

Stable Gelatin foam was prepared as described in section 3A.1 by making 250ml gelatin
solution for scale up stage 1 and 500ml gelatin solution for scale up stage 2. Mechanical
stirrer was used at agitation speed of 1400-1500rpm and whipping duration of 15 and 30
minute respectively. The foam produced was poured into trays and kept in a deep freezer
o o
for temperatures ranging from - 20 C to - 40 C. Foam was further dried in lyophilizer
o
by keeping drying temperature 50 C (less than Tg of gelatin) and applying vacuum.
Lyophilizer was designed to meet the capacity of scale up stages and Lyophilization
cycle was optimized.
Various process variables of Lyophilization process and desired end points for the final
product, gelatin sponge were determined on the basis of characteristics of the product
described in section 3A.3. All desired end points and their values are summarized in
table3.14.
Table 3.14 Lyophilization process variables and their effects on product characteristics
Variables Effects on product

End point determination Desired end point
Freezing temperature Pore size 80-100μm
Freezing time Wall thickness 4-5μm
Drying temperature Residual crosslinking agent conc. Less than 0.001%
Drying time In vitro biodegradation Less than 72 hrs
Water absorption 40-50 times
3A.4.3.2 Design of lyophilizer

After optimization of formulation process parameters, depending on the total water
content of the initial product, lyophilizer was selected for pilot scale and industrial scale
up. Water content of the developed gelatin sponge formulation was found to be 25%
w/w. On the basis of water content, condenser capacity of lyophilizer and vacuum pump
capacity was varied. Dimensions and specifications of lyophilizer are given in table 3.15.
Table 3.15 Specifications of lyophilizer used for scale up of gelatin sponge manufacturing process
Lyophilizer specifications for Lyophilizer specifications for Lyophilizer specifications for

Laboratory scale pilot scale Industrial scale
Basis: 5 pieces of 8 x5 x1cm size Basis: 20 pieces of 8 x5 x1cm size Basis: 60 pieces of 8 x5 x1cm size
Volume of solution= 50ml Volume of solution= 250ml Volume of solution= 500ml
Number of trays= 1 Number of trays= 3 Number of trays= 18
Condenser capacity: 0.5 litre Condenser capacity: 0.5 litre Condenser capacity: 0.8 litre
Vacuum pump capacity: Vacuum pump capacity: Vacuum pump capacity:
0.01mbar 0.01mbar 0.001mbar
Total surface area of the product Total surface area of the product Total surface area of the product
dried: 227cm2 dried: 720cm2 dried: 2100cm2
o
Since the drying temperature for the product was specified as below Tg [50 C], the
o
heating arrangement was required in the drying chamber of lyophilizer to achieve 50 C
temperature.
Alternate heating arrangement above and below the tray where the gelatin sponge is
placed could give uniform heating of the product. This would ultimately result in
uniform drying of the product. Design of drying chamber for Lyophilizer is explained
below in figure 3.14.
Front view Tray & heater arrangement

Heater plate
Tray
Drying chamber
Dimension of tray: 250x280mm

Total number of trays: 5
Heating plates: 6
Figure 3.14 Drying chamber design for industrial scale lyophilizer
Lyophilization cycle was optimized for scale up at both the steps of stage 1 and 2. On the
basis of the details summarized in table 3.14, various Lyophilization cycles were tried
and optimized as given in table 3.16.
Table 3.16 Optimization of Lyophilization cycle for Scale up for stage 1 and stage 2
Sr. Trial batch Lyophilization cycle Pore size of wet Pore size of dried
No. sponge (µm) sponge (µm)
o
1 LCT1 o 150-200 > 200
o
Heater temp: + 50 C
o
2 LCT2 o 100-125 150 -200
o
Heater temp: + 50 C
o
3 LCT3 o 80-100 100 - 120
o
Heater temp: + 50 C
o
4 LCT4 o 80-100 90 -110
o
Heater temp: + 50 C
o
5 LCT5 o 80-100 90-110
o
Heater temp: + 50 C
For both the scale up stages, Lyophilization trial LCT4 was found to give desired pore
o
size since to freezing at -35 C creates small ice crystals of size 90-110μm in wet sponge
which remained same after Lyophilization for dry sponge.
Gelatin foams produced at scale up stage 1 and stage 2 were dried by following the
Lyophilization trial LCT4 as given in table 3.16. The end points for the primary and
secondary drying were determined as given in table 3.17.
Table 3.17 Determination of end point of primary and secondary drying

[Thickness of sponge: 1cm]
Duration of Moisture content of gelatin sponge (%)
Lyophilization (hrs) Stage 1 Stage 2
6 40 50
10 25 27
14 15 14 Primary drying complete
16 8 7 Secondary drying complete
Vapor pressure: 2 mbar
Total drying of scale up batch of stage 1 and stage 2 was completed within 16 hrs
resulting in a dry gelatin sponge with moisture content of 8 and 7% respectively.
Scale up of Lyophilization process was successfully achieved with designing of

lyophilizer for scale up stage 1 and stage 2. This indicates commercial viability of the
formulation.
Selected scale up batches of gelatin sponge GSC2.2 and GSC3.3, given in table 3.13
were dried by optimizing the Lyophilization process as per the specification of trial
LCT4. The resulting gelatin sponges obtained were characterized as per procedures
described in section 3A.3. Results are reported in table 3.18
Table 3.18 Characterization of sponges of scale up batches
Sr. Tests Scale up Batch no.

no.
GSC2.2 GSC3.3
1. Appearance. White, soft White, soft

and porous and porous
2. Pore size(µm) 80-110 80-110
3. Wall thickness(µm) 4-5 4-5
4. Water absorption 40 - 50 40 - 50
5. Digestibility(min) 15 16
6. In vitro biodegradation (hrs) <72 <72
7. Residual Formaldehyde content <0.001% <0.001%
8. Sterility Sterile Sterile
After characterization and evaluation of developed sponges, the final product was packed
in double paper envelopes with polyethylene lining, heat sealed with heat sealing
machine. They were then packed in a carton and were sent for sterilization with γ
irradiation at ISOMED, Vadodara for radiation dose of 2.4Mrad.
Results and discussion

 Scale up of process for manufacturing of gelatin sponges was carried out at pilot
scale and commercial scale production on the basis of 20 pieces and 60 pieces of
8x5x1cm size of gelatin sponge per cycle. Scale up process was divided into
formulation process scale up and lyophilization process scale up.
 Formulation process was optimized as per the desired end points as given in table
3.14. Dimensional scale up method was used for design of vessel of
manufacturing for scale up. The dimensions of vessel and stirrer were increased
proportionately as per the size of product as given in table 3.12.
 The formulation process of gelatin sponge was optimized for whipping
conditions with varying gelatin concentration of 5% and 6% (Table 3.13). Stable
homogenous gelatin foams with small (80-120μm) size pores could only be
achieved from gelatin concentration of 5% w/v of gelatin solution at pH 7.5 and
at formaldehyde concentration of 0.3mg/ml at the whipping duration of 15
minutes for stage 1 of scale up and 30 minutes for stage 2 of scale up as reported
in table 3.11. This may be due to increased volume of gelatin solution which may
need more time for whipping for stable foam generation.
 Lyophilizer for scale up of the product was selected considering moisture content
of the product which was 25% of its weight. Specifications for laboratory scale,
pilot scale and commercial scale lyophilizer are given in table 3.15.
 Lyophilization process was optimized to get desired end point by optimizing
freezing temperature, freezing time, drying temperature and drying time. Freezing
temp of - 35oC, Condenser temp of - 50oC and Heater temp of + 50oC were found
to give white, soft gelatin sponge with pore size in the range of 60-100 μm, 0.2-
0.3 μm of wall thickness, 40-50 times water absorption. Same Lyophilization
cycle was applicable for both the scale up stages (table 3.13). Complete drying of
gelatin sponge at both the stages of scale up was occurred within 16hrs of
Lyophilization (Table 3.17).
 All the scale up batches showed good batch to batch reproducibility showing pore
size within 60-120µm. Water absorption was found to be 40-50 times the weight
of sponge. Digestion time for all sponges was less than 60 minutes. The in vitro
biodegradation of all formulations occurred within 72 hours. Residual
formaldehyde content was less than 0.001% indicating safe for use of gelatin
sponge.
 Final product gelatin sponge, was packed in double envelope and then in carton
to protect from environmental conditions and was subjected for gamma radiation
sterilization at the dose of 2.4Mrad. Final product were characterized and
checked for sterility. Results are given in table 3.18.
Conclusion
Thus this part of experimental work indicated that the developed procedure and process
will enable the production of absorbable gelatin sponges to commercial scale using
freeze drying. Process of freeze drying with modified freeze dryer is cost effective,
economical and resulted in development of biodegradable sponges similar to those
available in international market. Such sponges have potential application as
biodegradable haemostats in surgical procedures.
Chapter 3B Development of sustained release drug loaded absorbable gelatin sponges
Wound infections are one of the most frequent complications after surgery. All wounds
contain bacteria and even if the wound is healing normally, a limited amount of bacteria
will be present. But if the bacteria count rises, the wound may become infected. Bacterial
overload in a wound can lead to a serious infection that requires antibiotic treatment. In
surgical procedures, postoperative infection is poorly controlled and is widely considered
as a poorly met medical need [Kristl J, et.al; 1993]. Every year over 150 million people
undergo surgery and billions of dollors are spent on post-operative wound infection
management.
Following tissue injury, persistent inflammation triggers the release of mediators that
activate local pain receptors. This results in greater sensitivity of the surrounding skin and
deeper structures in the wound base. The pain itself can be caused by tissue damage
(nociceptive) [Gottrup et al; 2008] or nerve damage (neuropathic) [Woo et al; 2008]. Pain
has a protective function in nature, warning of damage, and promoting careful treatment
of the affected area [Goodwin SA; 1998]. Postoperative pain can be destructive by
heightening the cellular stress response, resulting in protein breakdown, platelet
aggregation, nausea, ileus and a suppressed immune system [Kehlet H; 1997]. Low
oxygen tension and poor perfusion can slow down the deposition of collagen in tissue
undergoing repair. Restricted breathing due to pain can lead to low-grade hypoxia, and
severe pain can cause vasoconstriction, both of which ultimately impair wound healing
[Jonsson K, et.al; 1991]. Persistent or chronic wound pain not only affects your patient’s
quality of life, it can also be a major barrier to wound healing.
Post-surgical infection is controlled with antibiotics but systemic administration of these

drugs can result in unfavourable side effects. Opiods such as morphine are used as pain
relieving agents for postoperative pain acting on opiod receptors in the brain and spinal
cord, but can also give rise to a range of adverse effects such as, in extreme cases,
respiratory depression, nausea, drowsiness etc [Thomas DR, et.al; 2000]. Such effects can
be minimized by providing antimicrobial substances in dressing itself and providing local
analgesia. Sponges are generally used for haemostatic activity; they can also function as a
device for safe and effective delivery of pharmaceuticals to targeted tissue at a controlled
rate. Gentamicin sulphate and lidocaine HCl were used as drug candidates to prevent
postoperative infection and postoperative pain.
Development of sustained release drug loaded absorbable gelatin sponges
Conventionally gelatin sponges are used as drug carriers by soaking the gelatin sponge in
respected drug solution. This may cause inappropriate dosing as well as immediate
action. Although gelatin sponges have very high elasticity and can be well hydrated
because of their high porosity and large surface area, the drugs implanted in or onto
sponge lamellae could only be retarded for a short period of time [Simamora PY, et.al;
1996].
In recent years, sponges based on natural biodegradable polymers have attracted much
attention for drug implantation as taking out the implant after treatment always causes
new damage to the wound. The patient comfort and optimum healing require soft and
elastic materials [Kristl J, et.al; 1993].
On this basis it seems promising to incorporate sustained release microspheres of

respective drugs into the sponge during formulation for sustained drug delivery. The
microspheres developed should be biodegradable to incorporate in biodegradable
sponges.
In the present study, microspheres of gentamicin sulphate and lidocaine HCl were
developed as model drugs to prepare the drug loaded surgical dressings for advanced
wound management. The objective of current work was to develop and characterize
biodegradable, sustained release microspheres in order to improve wound management.
Biodegradable microspheres
Encapsulation of drug molecules in particulate carriers as a method of controlled delivery
of molecules has been studied extensively [Watts PJ, et.al; 1990]. Biodegradable
microspheres are used to control drug release rates and to target drugs to specific sites in
the body, thereby optimizing their therapeutic response, decreasing toxic side effects, and
eliminating the inconvenience of repeated injections [Perrin DA, et.al; 1997].
Biodegradable microspheres have the advantage over large polymer implants in that they
do not require surgical procedures for implantation and removal. Gelatin and sodium
alginate are some of the biodegradable and biocompatible polymers that have
reproducible and slow-release characteristics [Anderson JM, et.al; 1997, Okada H, et.al;
1995].
Use of gelatin in pharmaceutics is particularly attractive by virtue of its biocompatibility
and biodegradability along with a total absence of toxicity or allergic problems. But
disadvantage of gelatin microspheres is that drug release rates are usually rapid;
therefore, these microspheres are not useful for long term controlled release [Tsung M,
et.al; 2001]. Consequently, gelatin and sodium alginate in combination may be useful to
design targetable controlled-release microsphere systems.
Being soluble polymers, they have to be chemically cross-linked to become insoluble at
37°C. Aldehyde derivatives such as formaldehyde, glutaraldehyde or other bifunctional
reactants have been used to produce insoluble biodegradable gelatin microspheres
[Raymond G, et.al; 1990].
Microspheres, in the present research project were prepared by emulsion chemical

crosslinking method. In this method, it is important to remove excess oil by washing the
particles with solvents such as isopropyl alcohol. Otherwise, the oil retained in the
microspheres may cause aggregation and alter the morphological properties of the
microspheres. This washing procedure is also to remove excess of the cross-linking agent
[Sahin S, et.al; 2002]. Isopropyl alcohol used to remove excess crosslinking agent also
extracts the water content of microspheres to obtain hardened microspheres that are easily
filtered and dried.
Microspheres of GS and LH were prepared separately and loaded into the absorbable
gelatin sponges to provide controlled drug release of respective drugs. Since the pore size
of gelatin sponge was found to be within range of 80-120μm; the particle size range of
microspheres should be within the range of 60-140μm for incorporation into the sponge.
Microspheres having particle size much smaller and much bigger will be difficult to
incorporate into the sponge.
3B.1 Development of biodegradable microspheres

The drugs, lidocaine HCl and gentamicin sulphate were encapsulated separately and
analysed respectively by the following mentioned method.
Microspheres of gelatin were prepared at laboratory scale by emulsion chemical
crosslinking method using an overhead stirrer (Remi equipments, Mumbai). Five ml of
gelatin solution (20% in water) was preheated to 80 °C and added drop-wise to 70 ml of
liquid paraffin containing 1% w/w Span 80 The biphasic system was stirred under
turbulent flow conditions using an overhead stirrer to form an w/o emulsion.
Formaldehyde (1ml) was added to the w/o emulsion as crosslinking agent. The dispersion
was mixed for various time intervals at an appropriate speed (1400-1500 rpm). The
microspheres were then washed and dehydrated 3 times with 20 ml of isopropyl alcohol
while stirring. Microspheres were collected by vacuum filtration and were allowed to dry
at room temperature (25 °C). If microspheres appeared stickier in nature, 20%v/v acetone
was used for dehydration of microspheres.
The microspheres were optimized for parameters like particle size, drug entrapment, drug
release and percent yield.
Flow chart for preparation of microspheres by emulsion chemical crosslinking method is
given in figure 3.15.
Drug + Polymer + Water at 60o C Liquid paraffin+Span 80
(Aqueous phase) (Oil phase)
Aqueous phase added to oil phase under vigorous stirring

(Equip: Overhead stirrer; duration: 15 min.)
w/o emulsion obtained
Cooled to 10 o C
Addition of crosslinking agent and stirring for 30 minutes
Dehydration of microspheres using IPA/ Acetone
Hardened Microspheres collected by vacuum filtration and air dried.
Figure 3.15 Formulation flow chart for emulsion chemical crosslinking method
3B.1.1 Preparation of Blank Microspheres

The morphology of the microspheres and their drug release profiles are influenced by the
method of preparation and the process parameters. Alteration of number of variables such
as ratio of aqueous to organic phase, alteration of the stirring speed, the nature and
concentration of the emulsifier used in the external phase, affect characteristics of
microspheres such as sphericity, particle size, appearance, yield, entrapment efficiency
and in vitro release.
In the present work, prior to developing GS and LH microspheres using gelatin, blank
microspheres by emulsion chemical crosslinking method were developed. They helped to
determine the effect of various process parameters on the selected pharmaceutical
characteristics of gelatin microspheres and to optimize the various process and product
parameters.
Procedure for preparation of blank microspheres

Formulations of blank microspheres using natural polymer gelatin with varying
concentrations of 0.25, 5 and 1% and formaldehyde from 0.1, 0.2, 0.3 and 0.4% were
prepared as per the flow chart in figure 3.15. Liquid paraffin, 100ml was used as oil
phase and 0.1% span80 was incorporated as emulsifier. Microspheres were prepared
using the general procedure outlined in section 3B.1 using overhead stirrer. Microspheres
were separated by vacuum filtration, hardened using 25ml of IPA and air dried at room
temperature.
The effect of change in concentration of gelatin and formaldehyde on percent yield,
particle size and appearance of the microspheres was studied. The final product was
transferred into a petri dish and dried at room temperature for 24 hrs. Microspheres were
stored in glass vials for further evaluation.
Various formulations of blank microspheres prepared are listed in tables 3.19.

Table 3.19 Batches of blank microspheres prepared using gelatin
Ingredients Formulation Nos.
(%) B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12
Gelatin 0.25 0.25 0.25 0.25 0.5 0.5 0.5 0.5 1 1 1 1
Formaldehyde 0.1 0.2 0.3 0.4 0.1 0.2 0.3 0.4 0.1 0.2 0.3 0.4
The blank microspheres were characterized for yield, appearance, particle size, shape,
surface characteristics and percent water content as described below. Results of
characterization and effect of speed of stirrer on particle size of microspheres is reported
in table 3.20.
3B.1.2 Evaluation of blank microspheres

1. Percent yield
The percent yield was calculated using the following formula:
weight of microspheres
% Yield = X 100
weight of drug + weight of polymer
2. Appearance
The microspheres were evaluated for their visual as well as microscopic appearance. A
small amount of the product was suspended on the slide in glycerin, covered with a cover
slip and observed under optical microscope under high power lens with 65x
magnification. Photomicrographs of the relevant batches of microspheres were taken

under the high power microscope equipment with a camera and are shown in figure 3.16
i. Shape and surface characteristics: The shape and surface morphology of the
microspheres were investigated using Scanning Electron Microscopy technique. The
SEM photomicrographs of blank microspheres are shown in figure 3.17.
ii. Particle size: The microspheres were observed under optical microscope, under high
power lens for preliminary assessment of size. The particle size was given as the
diameter in m.
iii. Percent water content: The moisture content of the microspheres was determined by
Karl Fischer titrimetry method. About 10 ml of dehydrated methanol was added to the
titration vessel and titrated to the electrometric endpoint with Karl Fischer reagent.
About 0.1 g microspheres were transferred to the vessel through the side arm closed
with the stopper. While the sample in the methanol was kept stirring magnetically, the
titration was continued to the electrometric endpoint. The volume of the reagent used
and its water equivalence factor was utilized to determine the percentage moisture
content in the microspheres.
Results of characterization are reported in table 3.20.

Table 3.20 Characteristics of blank microspheres prepared using gelatin
Batch Appearance % Particle size ( m) % Water
No. Yield Low rpm Medium rpm High rpm content
B1 Spherical, soft. 59.9 260-370 250-350 180-240 6.08
B2 Spherical, soft, dry. 70.8 270-370 250-350 150-230 4.86
B3 Spherical, hardened. 81.9 150-250 140-250 100-120 1.18
B5 Spherical, soft. 46.3 180-270 180-270 100-200 5.02
B6 Spherical, semi-dry. 45.8 180-280 180-280 150-200 4.98
B7 Spherical, hardened, dry. 87.1 240-340 230-340 80-120 1.53
B8 Spherical, hardened 82.3 250-350 240-350 120-210 1.96
B9 Non-spherical, aggregation 49.9 150-250 150-250 120-200 4.59
B10 Non-spherical, aggregation. 58.9 180-270 180-270 100-200 5.24
B11 Spherical, hardened 85.22 180-280 180-280 60-150 1.23
Formulation batches of B3, B7 and B11 were spherical, dry microspheres with % yield of
81.9, 87.1 and 85.22 which were higher than other formulations. Percent water content
was also less in these batches. Speed of overhead stirrer was observed to affect particle
size of microspheres. Increase in concentration of formaldehyde caused increase in
particle size. High speed of 1400-1500rpm resulted in smaller particle size within average
range of 100-200μm. Formulations of B3, B7 and B11 were taken further for
development of drug loaded microspheres at high speed of 1400-1500rpm.
Figure 3.16 Photomicrograph of blank microspheres (65x) [Formulation B7]
3B.1.3 Results and discussion

Development of blank microspheres using gelatin
 The emulsion chemical crosslinking technique proved to be simple and convenient
process for preparation of gelatin microspheres.
 Addition of 0.1 % span80 as emulsifier and 0.3 % formaldehyde as crosslinking agent
with varying gelatin concentration of 0.25, 0.5 and 1% and 25ml IPA as hardening
agent resulted in well-defined, spherical microspheres with no aggregation. Increase
in concentration of gelatin beyond 1% resulted in aggregation of microspheres.
 Formulation of blank microsphere batches B3, B7 and B11 were found to give
spherical, dry microspheres with % yield of 81.9, 87.1 and 85.22 which were higher
than other formulations. Percent water content was also found to be less in these
batches. Speed of overhead stirrer (rpm) was found to affect particle size of
microspheres. Photomicrograph of blank microspheres revealed spherical
microspheres in the size range of 100-200μm without aggregation.
 High speeds rotation of stirrer of 1400-1500rpm was smaller particle size within
range of 100-250μm. This could be because of more efficient stirring of the emulsion
during preparation of microspheres.
Combination of formulations B3, B7 and B11 were used further for formulation of drug
loaded microspheres.
3B.2 Preparation and Evaluation of Drug loaded Microspheres

For evaluation of drug loaded microspheres, analytical method needs to be developed for
estimation of drug content and in vitro release profiles. Analytical methods for
estimation of GS and LH contents were developed by eliminating interference of
excipients.
3B.2.1 Analytical method development for estimation of drug content and in vitro
release profile
The λmax of GS for colorimetric estimation was found to be 324 nm and λmax of LH
was found to be 263 nm respectively (Section 2B). Developed spectroscopic methods
under chapter 2B for analysis of GS and LH were also validated for non-interference of
excipients for utilizing the same for estimation of drug content in developed formulation.
Non-interference of excipients
This study was carried out in order to find out interference of excipients with developed
analytical methods. Accurately weighed 1g of gelatin and 0.3g of formaldehyde were
dispersed in phosphate buffer pH 7.0 separately and filtered.
For GS microspheres: The resulting filtrate of each of the excipients was treated as per
the procedures mentioned in section 2B under colorimetric analysis of GS and scanned in
spectrum mode over range of 200-400nm and the absorbance at 324 nm was noted.
For LH microspheres: The resulting filtrate of each of the excipients was scanned in
spectrum mode over range of 200-400nm and the absorbance at 263 nm was noted.
Placebo batch was prepared, subjected to similar test and observed for absorbance at 324
nm and 263 nm.
No additional peak was observed for any excipients used in formulation development
process in the spectrum over range of 200-400nm. Absorbance values of both the placebo
formulation were found to be less than 0.005. Thus it was concluded that the selected
excipients did not interfere with the analytical procedure and selected spectroscopic
method was found to be satisfactory for determination of drug content and drug release
profiles of the developed formulations.
1. Drug content and Percent drug entrapment

Drug entrapment of microspheres was calculated in phosphate buffered saline pH 7.6.
Weighed amount of microspheres were suspended in the 100ml of medium under stirring.
Five ml of sample was withdrawn after 1hr and analysed with proper dilutions by
respective method of analysis for each drug. The experiments were conducted in
triplicate and the average value of the three readings was taken. The concentration of 1st
reading was used to predict the amount of unentrapped drug. Remaining microspheres in
the medium were kept in the medium and amount of drug released after 24 hours was
analysed for the amount of the drug present in microspheres. No more drug release was
observed after 24 hours.
Percent drug entrapment was calculated by following formula.
%drug content observed - % drug unentrapped

% drug entrapment = X 100
Theoretical drug content
2. In vitro release studies on microspheres

The release of incorporated drug from microspheres based systems occurs in two phases:
▪ The initial release of surface drug, described as “burst effect”, which is significant
due to the small particle size and large surface area of the dosage form.
▪ This is followed by sustained release of drug molecules from a matrix type
delivery system due to diffusion of drug from the polymer.
Release due to diffusion of drug molecules from the insoluble polymer follows either
zero order kinetics, first order kinetics or kinetics derived by Higuchi for diffusional
release from the drug-polymer matrices [Chuo W, et.al; 1996].
In the present work, the selected optimized formulations of GS and LH microspheres
based on complete evaluation of physicochemical properties were subjected to in vitro
release test to study the release profile of the drug from the microspheres.
Colorimetric method was used to measure the amount of Gentamicin sulphate at 324 nm
and UV Spectrophotometric method was used for determination of Lidocaine HCl at
263nm. Mechanism of drug release was determined using various kinetic models.
3B.2.2 Preparation of gelatin: drug loaded microspheres

Procedure
Formulation development of GS and LH loaded microspheres was carried out by
emulsion chemical crosslinking method using gelatin as per the flow chart in figure 3.15,
using the general procedure outlined in section 3B.1. Concentrations of gelatin and other
excipients used in combinations of B3, B7 and B11 were used for preparation.
Formulations were optimized for gelatin concentration by varying from 0.25-1g and drug
concentration from 100-500mg. Compositions for GS microspheres are given in table

3.21a and composition for LH microspheres are given in table 3.21b.
Optimization of process parameters
Percent yield and drug entrapments of GS and LH microspheres each were optimized by
22 factorial design using concentration of drug and polymer as process variables. Surface
response curves were plotted using Stat-Ease Design-Expert v.7 software [Figure 3.17;
3.18].
For GS microspheres:
Formulation parameters Lower level Higher level
Gelatin (g) 0.25 1
Gentamicin sulphate (mg) 100 500
For LH microspheres:
Formulation parameters Lower level Higher level
Gelatin (g) 0.25 1
Lidocaine HCl(mg) 100 500
Table 3.21a Composition of GS: gelatin microspheres

Formulation Gelatin GS % yield Entrapped
code (g) (mg) Drug (%)
GSM1 0.25 100 87 48
GSM1.1 0.5 100 88 52
GSM1.2 1 100 87.5 58
GSM2 0.25 250 87.6 43
GSM2.1 0.5 250 89 55
GSM2.2 1 250 89 60
GSM3 0.25 500 88.2 60
GSM3.1 0.5 500 87.8 64
GSM3.2 1 500 88.5 67
Table 3.21b Composition of LH: gelatin microspheres

Formulation Gelatin LH % yield Entrapped
code (g) (mg) Drug (%)
LHM1 0.25 100 89 48
LHM1.1 0.5 100 87 62
LHM1.2 1 100 88 64
LHM2 0.25 250 88.2 43
LHM2.1 0.5 250 87.5 55
LHM2.2 1 250 88.9 65
LHM3 0.25 500 88.7 67
LHM3.1 0.5 500 87 69
LHM3.2 1 500 88 72
Effect of drug and polymer concentration on % entrapment and % yield was studied by
equations obtained using surface response curves as shown in figures 3.17 and 3.18 and
Percent contribution of gelatin and drug concentration on % entrapment and % yield for
GS and LH microspheres was reported.
Optimization of GS microspheres
Design-Ease® Software
% yield
88.5
87
88.5
X1 = A: Gelatin
X2 = B: GS
88.125
% yield
87.75
87.375
87
500 1
400 0.8125
300 0.625
200 0.4375
B: GS A: Gelatin
100 0.25
Figure 3.17a Effect of gelatin and drug concentration on % yield of GS microspheres

Log10(% yield) = + 86.51667+0.73333× gelatin +3.1666 ×GS – 6.6666 × gelatin × GS
% entrapment
67
48
67
X1 = A: Gelatin
X2 = B: GS
62.25
% entrapment
57.5
52.75
48
500 1
400 0.8125
300 0.625
200 0.4375
B: GS A: Gelatin
100 0.25
Figure 3.17b Effect of gelatin and drug concentration on % entrapment of GS microspheres

Log10(% Drug entrapment) = + 36.16667+28.33333 × gelatin +0.065000 × GS - 0.07000 × gelatin × GS
Percent contribution of factors for GS microspheres

Factor % contribution
Drug Gelatin Both
% yield 11.59 87.68 0.72
% entrapment 33.96 14.22 51.82
Optimization of LH microspheres
% yield
89
88
89.1
X1 = A: Gelatin
X2 = B: LH
88.825
% yield
88.55
88.275
88
500 1
400 0.8125
300 0.625
200 0.4375
B: LH A: Gelatin
100 0.25
Figure 3.18a Effect of gelatin and drug concentration on % yield of LH microspheres

Log10(% yield) = + 89.43333-1.43333 × gelatin -1.000 × LH +1.000 × gelatin × LH
% entrapment
67
48
67
X1 = A: Gelatin
X2 = B: LH
62.25
% entrapment
57.5
52.75
48
500 1
400 0.8125
300 0.625
200 0.4375
B: LH A: Gelatin
100 0.25
Figure 3.18b Effect of gelatin and drug concentration on % entrapment of LH microspheres

Log10(% Drug entrapment) = + 38.16667+26.33333 × gelatin +0.063000 × LH - 0.07000 × gelatin × LH
Percent contribution of factors for LH microspheres
LH Gelatin Both
% yield 2.93 94.14 2.93
% entrapment 35.96 14.20 51.84
Formulations GSM3.2 and LHM3.2 showing higher entrapment of 67% of GS and 72%
of LH were taken further for characterization.
3B.2.3 Characterization and evaluation of microspheres

Characterization and evaluation of optimized drug loaded microspheres was carried out
as per the following methods.
1. Surface morphology
The surface morphology of microspheres was observed by Scanning Electron Microscope
(SEM). It was investigated using a Jeol Analytical Scanning Microscope, Model JSM-
840A/WDS/EDS System. The samples for SEM analysis were prepared by sprinkling the
dried microspheres powder onto one side of a double adhesive tape which was stuck to an
aluminium stub. The stubs were then coated with gold using an Edwards E-306 sputter
coater to a thickness of 20-30nm. The samples were then examined using SEM and their
photomicrographs were taken by the attached camera.
For both GS and LH, spherical particles were obtained with diameter ranging from 60-
150μm, having similar particle morphology and size. Photomicrographs and SEM images
are shown in figure 3.20 and 3.21 and results are reported in table 3.19 for GS
microspheres and table 3.20 for LH microspheres respectively.
2. Mean particle size and particle size distribution

Particle size analysis of microspheres was performed by optical microscopy using a
compound microscope. A small amount of dry microspheres was spread on a clean glass
slide. The slide containing microspheres was mounted on the stage and diameter of 200
particles was measured using a calibrated ocular micrometer and mean particle size was
determined. Results are reported in table 3.19 for GS microspheres and table 3.20 for LH
microspheres.
3. Moisture content
Moisture content of the microspheres was determined by using Karl Fischer moisture
determination apparatus (Veego/Matic-MD-PC model) as described under section 3B.2.1.
Results are reported in table 3.19 for GS microspheres and table 3.20 for LH
microspheres.
4. Determination of the swelling ratio

Developed microspheres of GS and LH were incorporated into biodegradable sponges
for sustained drug delivery. Since the swelling behaviour is an important factor of
developed sponges to be effective, swelling behaviour of microspheres to be
incorporated was also determined.
The swelling ratio of gelatin microspheres was determined by using distilled water as the
swelling medium. Microspheres were left in swelling medium for at least 60 min to reach
maximum swelling. Volumetric measurements were made by measuring the diameters of
microspheres placed in the swelling medium at appropriate time intervals using optical
microscopy. The swelling ratio was calculated from the ratio of the volume of swollen
particles to that of dry particles. Optical micrographs of swollen microspheres for GS are
shown in figure 3.20b and for LH in figure 3.21b. Results of swelling ratio obtained are
reported in table 3.20a and 3.20b for GS and LH microspheres respectively.
5. Drug content and Percent drug entrapment

Drug content and drug entrapment were calculated as per the methods described under
section 3B.2.1.
Results for percent entrapment for GS and LH microspheres are reported in tables 3.22a
and 3.22b.
6. In vitro drug release studies of microspheres

In vitro drug release profiles from microspheres were studied using Flow through cell
apparatus-USP IV (Figure 3.19). Flow through cell dissolution apparatus is used for
microspheres and have provided exciting results and a solution to troubles associated
with traditional dissolution methods. The assembly consists of a reservoir containing
release medium, a pump that forces the release medium upwords through the vertically
through the vertically positioned flow-through cell and a water bath. The pump usually
has flow rates of 4, 8 and 6ml/min. the bottom cone of the cell is filled with small glass
beads of about 1mm diameter and with one bead of about 5mm diameter positioned at the
apex to protect the fluid entry tube, whereas filter (glass fiber filter/ Whatmann filter) is
positioned at the inner top of the cell. Flow through cell apparatus can be operated as
closed system by recycling a fixed volume of the medium. The medium passes the
sample and is returned by the pump to the flow through cell and the sample. A reservoir
is placed in the line allowing the medium to be stirred, heated and sampled. By
determining the concentration of analyte and the volume in the system, the cumulative
release can be directly calculated. The versatility of the apparatus with respect to
alteration of flow rates can be used for studying the release characteristics from the
dosage form. The method is more reliable, reproducible and discriminative than other
systems.
In vitro release profile of microspheres was studied using Flow through cell apparatus
o
USP IV at 37 C with flow rate of 16ml/min (in 250ml phosphate buffer, pH7.4). Five
milliliters samples were taken, filtered through a 0.45μm filter and replaced with 5ml
o
fresh medium at 37 C. Samples were analysed by respective methods mentioned in
section 3B.2.1.
The release mechanism of GS and LH loaded microspheres was investigated based on the
plots of percent drug release versus time when the release of drug was sustained beyond
6hrs. The resulting data was fitted the release data into models representing
Zero order - percent drug release vs. time
First order - log percent retained vs. time
Higuchi - percent drug release vs. square root of time
Figure 3.19 Flow through cell for in vitro release of microspheres
Drug release studies were carried out on formulation GSM3.2 of GS microspheres and
LHM3.2 of LH microspheres. Percent drug release versus time for gelatin: GS
microspheres and gelatin: LH microspheres are shown in figures 3.22A and 3.22B
respectively.
7. Digestibility of microspheres
Developed sustained release microspheres are to be incorporated into gelatin sponge
which gets absorbed inside the body and digested with the proteolytic enzymes at the site.
The digestibility of developed microspheres was checked by following the procedure
mentioned in section 3A.3. Results are given in table 3.22a for GS microspheres and
3.22b for LH microspheres.

Analytical methods for estimation of GS and LH contents were developed by eliminating
non-interference of excipients for evaluation of drug content and drug release from
developed formulations of microspheres.
Analytical method development for estimation of drug content and in-vitro release
profile
Developed spectroscopic methods under chapter 2B for analysis of GS and LH were
validated for non-interference of excipients by evaluating UV absorbance for excipients.
No additional peak was observed for any excipients used in formulation development
process in the spectrum over range of 200-400nm. Absorbance values of both the placebo
formulation were found to be less than 0.005. Selected excipients did not interfere with
the analytical procedure and selected spectroscopic method was found to be satisfactory
for determination of drug content and drug release profiles of the developed formulations.

 Drug loaded microspheres of gelatin were prepared with drug: polymer ratio as
shown in table 3.18a for GS microspheres and 3.18b for LH microspheres.
Contributions of polymer and drug concentrations on percent yield and entrapment
efficacy of microspheres were studied by plotting surface response curve by Stat-
design-ease 7 software. Surface response curves are shown in figure 3.17a and 3.17b.
 For GS microspheres, percent contributions of gelatin, GS and both the factors on %
yield were observed to be 87.68, 11.59 and 0.72 and for LH microspheres, they were
found to be 94.14, 2.93 and 2.93 respectively. Contribution of gelatin was 94.14%
which was higher than other factors. This suggested that % yield of both the
microspheres was dependent only upon gelatin concentration (Table 3.19a; 3.19b).
 For GS microspheres, percent contribution of gelatin, GS and both the factors on %
entrapment was observed to be 33.96, 14.22 and 51.82 and for LH microspheres, it
was found to be 35.96, 12.20 and 51.84. These values suggested that % entrapment of
both the microspheres was found to be more dependent upon collective concentration
of gelatin and drug used (Table 3.19a; 3.19b).
 For GS microspheres, drug entrapment was found to be higher (67%) in case of
formulation GSM3.2 having composition of 1g gelatin and 500mg of GS compared to
other formulations. In case of LH microspheres, drug entrapment was found to be
higher (72%) for formulation LHM3.2 containing 1g gelatin and 500mg of LH,
compared to other formulations. Percent yield of GS microspheres was 88.66±1.64
and for LH microspheres, it was 88.78±1.2 as given in tables 3.21a and 3.21b.
Formulations GSM3.2 and LHM3.2 showing higher entrapment of 67% of GS and 72%
of LH were taken further for characterization.
Characterization and evaluation of developed microspheres

Formulations GSM3.2 and LHM3.2 of GS and LH microspheres were characterized for
morphology using optical microscope and SEM. Photomicrographs of microspheres,
swollen microspheres, SEM images and particle size distribution graphs of GS and LH
microspheres are shown in figure 3.20 and 3.21.
a. GS microspheres (65x) b. GS loaded swollen microspheres (65x)
120
250-300
100
Frequency
80
60 300-350
40
50-100
20 200-250 400-450
100-150
0
0 2 4
Particle size (μm)6 8
c. SEM image (200x) d. Particle size distribution

Figure 3.20 Morphology of GS microspheres
Scanning electron microscopy revealed very large particle size distribution of 250-300μm
with mean particle size of 298± 0.2μm for GS microspheres. Swelling ratio for GS
microspheres was found to be 8.2± 0.15
a. LH microspheres (65x) b. LH loaded swollen microspheres (65x)
100
90 300-350
80
70
Frequency
60 250-300
50
40
30
20 50-100 200-250
10 100-150 400-450
0
0 2 4 6 8
Particle size (μm)
Figure 3.21 Morphology of LH microspheres
Scanning electron microscopy revealed particle size distribution of 300-350μm with

mean particle size of 293.24 ± 10.5μm for LH microspheres (Figure 3.20c; 3.21c).
Swelling ratio for LH microspheres, it was found to be 3.2± 0.15.
3B.3.4 In vitro release studies on microspheres

In vitro release profiles of GS and LH microspheres were investigated using Flow
through cell apparatus USP IV using Phosphate buffer; pH 7.4. Figures 3.23a and 3.23b
illustrates the in vitro release of GS and LH from gelatin microspheres respectively.
120
100
80
% drug released
60
40
y = 45.95x + 20.60
20 R² = 0.824
0
0 0.5 1 1.5 2 2.5
Time (hrs)
a. Percent GS release Vs time (hrs)

Figure 3.22a In-vitro drug release kinetics Gelatin: GS microspheres
120
100
% drug released
80
60
40 y = 20.38x + 29.56
20 R² = 0.704
0
0 1 2 3 4 5
Time (hrs)
a. Percent LH release Vs time (hrs)
Figure 3.22B In-vitro drug release kinetics of Gelatin: LH microspheres
GS and LH microspheres failed to sustain the drug release more that 4 and 3 hrs.
Results of characterization for selected formulations of GS and LH microspheres are
given in table 3.22a and 3.22b.
Table 3.22a Results of evaluation of GS: gelatin microspheres [Formulation: GSM3.2]
Sr. No. Parameters Observations
1 Physical appearance and surface Free flowing, pale yellow coloured, individual,
morphology spheres with uneven surface.
2 Yield %w/w 88.66 ± 1.64
3 Moisture content %w/w 5.65 ± 0.05
4 Mean particle size (µm) 298± 0.2
5 Particle size distribution (µm) 280-320
6 Swelling ratio 8.2± 0.15
7 Entrapment efficiency %w/w 67± 0.56
8 In-vitro release profile Non-linear release within 2 hrs.
9 Digestibility < 25 min.
Table 3.22b Results of evaluation of LH: gelatin microspheres [Formulation: LHM3.2]

1 Physical appearance and surface Free flowing, off white coloured, individual,
morphology smooth surfaced spheres
2 Yield %w/w 88.78 ± 1.2
4 Mean particle size (µm) 293.24 ± 10.5
7 Entrapment efficiency %w/w 72 ± 0.67
8 In-vitro release profile Non-linear release within 4 hrs.
GS and LH loaded gelatin microspheres were found to be of very large size (average=
300μm), as well as failed to give sustain the release which is not preferable for the
incorporation into gelatin sponge. Hence, gelatin along with another natural,
biodegradable polymer, sodium alginate in combination was therefore tried to sustain
drug release. This procedure is described in next section 3B.2.
3B.3 Formulation development of gelatin: sodium alginate microspheres

Gelatin, being highly soluble and biodegradable natural polymer, was not able to sustain
the release of drugs GS and LH more than 4 hrs. Thus realizing the necessity for
achieving predetermined and sustained release applications of GS and LH for advanced
wound management, crosslinked microspheres were designed using gelatin in
combination with another natural biodegradable polymer sodium alginate [Soni ML,
et.al; 2010, Takka S, et.al; 1999]. Suitability of developed microspheres was evaluated by
characterizing in terms of drug content and drug release profile as discussed in section
3B.2.
3B.3.1 Preparation of gelatin: sodium alginate drug loaded microspheres

Procedure
Formulations of GS and LH loaded microspheres prepared by emulsion chemical
crosslinking method using gelatin and sodium alginate as polymers. As per the flow chart
in figure 3.15, the general procedure outlined in section 3B.1 was used for preparation of
microspheres. Blank microsphere formulation combinations of B3, B7 and B11 were
utilized for drug loading. Formulations were optimized for gelatin: sodium alginate ratio
by varying gelatin concentrations from 0.25-1g, sodium alginate concentration of 0.5-1g
and drug concentration of 100-500mg. Compositions along with data for % yield and %
entrapment of GS in microspheres are given in table 3.23a and the results for LH
microspheres are given in table 3.23b.

Percent yield and drug entrapment of GS and LH microspheres each were optimized by
22 factorial design using concentration of drug and ratio of gelatin and sodium alginate
polymer as formulation variables. Surface response curves were plotted using Stat-Ease
Design-Expert v.7 software.
For GS microspheres:
Process parameters Lower level Higher level
Gelatin: Sodium alginate (g) 0.25:0.5 0.25:1
Gentamicin sulphate (mg) 100 500
For LH microspheres:
Process parameters Lower level Higher level
Gelatin: Sodium alginate (g) 0.25:0.5 0.25:1
Lidocaine HCl(mg) 100 500
Effect of drug and gelatin: sodium alginate ratio on % entrapment and % yield was
studied by equations obtained using surface response curves as shown in figures 3.23a
and 3.23b.
Table 3.23a Composition of GS: gelatin: sodium alginate microspheres
Formulation Gelatin Sodium GS % yield Entrapped
code (g) Alginate (mg) Drug (%)
(g)
GAM1 0.25 1 100 88 58
GAM1.1 0.25 0.5 89 60
GAM2 0.5 1 100 89 54
GAM2.1 0.5 0.5 90 57
GAM3 1 1 100 87 51
GAM3.1 1 0.5 88 55
GAM4 0.25 1 250 86 62
GAM4.1 0.25 0.5 88 64
GAM5 0.5 1 250 88 60
GAM5.1 0.5 0.5 91 65
GAM6 1 1 250 88 56
GAM6.1 1 0.5 91 59
GAM7 0.25 1 500 87.6 60
GAM7.1 0.25 0.5 88.1 64
GAM8 0.5 1 500 86.9 64
GAM8.1 0.5 0.5 87.9 66
GAM9 1 1 500 88 67
GAM9.1 1 0.5 90 70
GAM10 1 1 600 87 65
GAM10.1 1 0.5 88 66
Table 3.23b Composition of LH: gelatin: sodium alginate microspheres

Formulation Gelatin Sodium GS % yield Entrapped
code (g) Alginate (mg) Drug (%)
(g)
LAM1 0.25 1 100 88 62
LAM1.1 0.25 0.5 90 65
LAM2 0.5 1 100 89 60
LAM2.1 0.5 0.5 91 64
LAM3 1 1 100 87 58
LAM3.1 1 0.5 90 61
LAM4 0.25 1 250 86 43
LAM4.1 0.25 0.5 88 48
LAM5 0.5 1 250 88 55
LAM5.1 0.5 0.5 91 59
LAM6 1 1 250 88 65
LAM6.1 1 0.5 90 69
LAM7 0.25 1 500 87.6 67
LAM7.1 0.25 0.5 88.9 69
LAM8 0.5 1 500 86.9 69
LAM8.1 0.5 0.5 90 72
LAM9 1 1 500 88 72
LAM9.1 1 0.5 91 76
LAM10 1 1 600 86 68
LAM10.1 1 0.5 90 70
Optimization of GS microspheres
% yield
90
86.9
90
X1 = A: Alginate
X2 = B: GS
89.2
% yield
88.4
87.6
86.8
500.00 1.00
400.00 0.88
300.00 0.75
200.00 0.63
B: GS A: Alginate
100.00 0.50
Figure 3.23a Effect of sodium alginate and drug concentration on % yield of GS microspheres
Log10(% yield) = + 92.8000-6.50000 × alginate- 0.018000 × GS+0.025500 × alginate × GS
% entrapment
70
51
70
X1 = A: Alginate
X2 = B: GS
65.25
% entrapment
60.5
55.75
51
500 1
400 0.875
300 0.75
200 0.625
B: GS A: Alginate
100 0.5
Figure 3.23b Effect of sodium alginate and drug concentration on % entrapment of GS microspheres
Log10(% Drug entrapment) = + 56.7500-10.50000 × alginate+2.5000 × GS+0.045000 × alginate × GS
Percent contribution of factors for GS microspheres

GS Alginate Both
% yield 2.89 4.32 92.79
% entrapment 90.33 0.97 8.70
Optimization of LH microspheres
% yield
91
86.9
91
X1 = A: Alginate
X2 = B: LH
89.975
% yield
88.95
87.925
86.9
500 1
400 0.875
300 0.75
200 0.625
B: LH A: Alginate
100 0.5
Figure 3.24a Effect of gelatin and drug concentration on % yield of LH microspheres

Log10(% yield) = + 93.05000-7.050000 × alginate- 0.020000 × LH+0.030500 × alginate × LH
% entrapment
76
58
76
X1 = A: Alginate
X2 = B: LH
71.5
% entrapment
67
62.5
58
500 1
400 0.875
300 0.75
200 0.625
B: LH A: Alginate
100 0.5
Figure 3.24b Effect of gelatin and drug concentration on % entrapment of LH microspheres

Log10(% Drug entrapment) = + 62.000-8.50000 × alginate-9.8534 × LH+0.045000 × alginate × LH
Percent contribution of factors for LH microspheres

LH Alginate Both
% yield 7.98 9.75 82.27
% entrapment 87.31 2.99 9.70
From the results obtained from the surface response curve methodology, it was observed
that concentration of sodium alginate alone did not have major effect on percent yield and
drug entrapment of both GS and LH microspheres. Increase in concentration of drug
resulted in drug entrapment of microspheres upto 500mg of drug concentration. Further
increase in drug concentration caused reduction in drug entrapment. Formultions

GAM9.1 and LAM9.1 which showed drug entrapment of 70% and 78% for GS and LH
microspheres were further characterized.
3B.3.2 Characterization and evaluation of drug loaded microspheres

Characterization of developed microspheres was carried out for following parameters as
desribed in section 3B.2.3.
1. Surface morphology: (Figure 3.25;3.26)
2. Mean particle size and particle size distribution
3. Moisture content
4. Determination of swelling ratio.
5. Drug content and percent entrapment
6. In vitro drug release
Photomicrographs and SEM images of prepared microspheres, swollen microspheres,
SEM images of formulation and particle size distribution graph of GAM9.1 of GS
microspheres and LAM9.1 of LH microspheres are shown in figure 3.25 and 3.26.
Moisture content, swelling ratio, drug content and percent entrapment of GS and LH
microspheres are reported in table 3.28a; 3.28b.
Selected formulations of microspheres were further characterized for drug content by
FTIR spectroscopy and residual solvent analysis for determination of concentration of
residual solvents in prepared microspheres as described below.
Drug entrapments by Fourier transform infrared microscopy

Infrared spectra were recorded for formulation GAM9.1 and formulation LAM9.1, with
FTIR spectrometer to confirm GS and LH entrapment in the microspheres. Samples were
prepared by processing compressed KBr disks. Frequencies for drug and polymer in
formulation were observed and recorded in figure 3.27a for GS microspheres and figure
3.27b for LH microspheres.
In vitro drug release

In vitro drug release profiles and values of t50% are given in figure 3.28; table 3.24 for GS
microspheres and figure 3.29; table 3.25 for LH microspheres.
Residual solvents by Gas Chromatography

The developed microspheres are incorporated into the sponges for sustained drug
delivery so they should be analysed for residual solvent content to avoid toxic effects of
solvents in the system. Formulation GAM9.1 and LAM9.1 were analyzed for residual
solvents content by method described below.
Isopropyl alcohol and acetone were utilized as solvents for formulation of microspheres.
The analysis for residual isopropyl alcohol and acetone content was carried out using gas
chromatography headspace injection method.
Preparation of samples
Standard mixture -Isopropyl alcohol (50μg/ml) and acetone (50μg/ml) were diluted
to10ml with dimethyl formamide.
Microsphere sample - 100mg of microsphere sample was accurately weighed and 10ml
of dimethyl formamide was added. The suspension was sonicated and used for injection.
Procedure for analysis of residual solvents

The microsphere sample solution (2ml) and standard mixture solution (2ml) were placed
in a 20ml vial and sealed separately. The vials were incubated at 105oC with agitation for
20min and 900µl of headspace was injected into the chromatographic system. The
chromatograms were recorded and peak area corresponding to isopropyl alcohol and
acetone was measured. The analysis was carried out in triplicates and average or three
determinations were taken. The results of gas chromatographic studies are given in figure
3.30 and table 3.27.

Preparation of gelatin: sodium alginate drug loaded microspheres
Microspheres of GS and LH were prepared using gelatin along with sodium
alginate for retarding the drug release. The compositions tried for formulation of
GS and LH microspheres are given in tables 3.23a and 3.23b. Gelatin was tried in
the varying concentrations from 0.25 to 1g along with sodium alginate with
varying concentration of 0.5 and 1g. Various ratios of polymers were tried and
evaluated for % yield and entrapment.
For GS microspheres, formulation GAM9.1 showed drug entrapment of 70% and
for LH microspheres, formulation LAM9.1 showed drug entrapment of 76% with
gelatin: sodium alginate ratio of 1:0.5 and drug concentration of 500mg which
was higher than other formulations [Table 3.23a; 3.23b]. Further increase in drug
concentrations in both the cases did not increase the drug content significantly and
further increase in gelatin: alginate ratios formed the aggregates of microspheres.
Method optimization
Sodium alginate contribution for % yield and entrapment was studied by plotting
surface response curve by Stat-design-ease 7 software. Surface response curves
and values for contribution are shown in figure 3.23a, 3.23b for GS microspheres
and in figures 3.24a, 3.24b for LH microspheres.
Sodium alginate alone has contributes to the extent of 4.32% and 0.97% for GS
microspheres whereas 9.75% and 2.99% for LH microspheres on % yield and
entrapment. Whereas concentration of GS and LH were found to contribute to the
extent of 90.33% and 87.31% towards drug entrapment. Combination of gelatin:
sodium alginate increased % entrapment of drug.
Characterization and evaluation of developed microspheres

Formulations GAM9.1 and LAM9.1 of GS and LH microspheres were characterized for
morphology using optical microscope and SEM. Photomicrographs of microspheres,
swollen microspheres, SEM images and particle size distribution graphs of GS and LH
microspheres are shown in figure 3.25 and 3.26.
a. GS microspheres (65x) b. GS loaded swollen microspheres (65x)
90
80 80-100
70
Frequency
60 100-120
50
40
30
20
20-40 60-80 120-140
10 40-60
0
0 2 4 6 8
Particle size (μm)
Figure 3.25 Morphology of Alginate: gelatin: GS microspheres [Formulation: GAM9.1]
a. LH microspheres (65x) b. LH loaded swollen microspheres (65x)
90
80 100-120
70
Frequency
60 80-100
50
40
30
20 20-40 60-80 120-140
10 40-60
0
0 2 4
Particle size (μm)6 8

Figure 3.26 Morphology of Alginate: gelatin: LH microspheres [Formulation: LAM9.1]
GS microspheres were found to be spherical, uniform, free flowing and pale

yellow in color which may be due to color of pure drug whereas LH microspheres
were also found to be spherical, uniform, free flowing and white in color as that
of pure LH [Figure3.25; 3.26].
SEM revealed mean particle size 90.52±2.4μm, with particle size distribution of
80-100μm for GS microspheres and mean particle size of 110.32±3.4μm, with
particle size distribution of 100-120μm for LH microspheres prepared using
gelatin: sodium alginate combination. This showed reduction in particle size was
observed than that of microspheres prepared with gelatin alone that may be due to
incorporation of sodium alginate.
Swelling ratio for GS microspheres was found to be 10.2± 0.1 and for LH
microspheres, it was found to be 9.2± 0.1 indicating uniform swelling which was
more than that of only gelatin microspheres.
Drug entrapments by Fourier transform infrared microscopy

Infrared spectra were recorded for formulation GAM9.1 and formulation LAM9.1, with
FTIR spectrometer to confirm GS and LH entrapment in the microspheres.
FTIR spectra of GS microspheres

3527cm-1: OH groups
1691cm-1: amide groups of GS indicating drug entrapment
1263cm-1: C-O-C groups
Figure 3.27a FTIR analysis of GS microspheres for drug entrapment [Formulation GAM9.1]
FTIR spectra of LH microspheres

3649cm : OH groups, 1697cm-1: R-CO-NH-R groups
-1
1234 cm-1: CN groups, 786 cm-1: aromatic ring

Figure 3.27b FTIR analysis of LH microspheres for drug entrapment [Formulation: LAM9.1]
FTIR Spectra for GS and LH microspheres showed presence of GS at 1691cm-1, 1234

cm-1 and LH groups at 1697cm-1,1234 cm-1 and 786 cm-1 wavelength indicated drug
entrapment.
In vitro release studies on microspheres

In vitro release profiles of GS and LH microspheres were investigated using Flow
through cell apparatus USP IV using Phosphate buffer; pH 7.4. Figures 3.28 and 3.29
illustrated the in vitro release of GS and LH from gelatin microspheres respectively.
Regression parameters of different release kinetic models are given in table 3.24 and
3.25.
120
100 y = 11.58x + 11.57
% drug released
R² = 0.968
80
60
40
20
0
0 2 4Time (hrs)6 8 10
a. Percent gentamicin sulphate release Vs time (hrs)

Zero order release of GS
2.5
y = -0.195x + 2.138
log (% drug retained)
2 R² = 0.892
1.5
0.5
0
0 2 4 6 8 10
Time (hrs)
b. Log percent drug retained Vs time (hrs)

First order release
120
y = 35.22x - 5.190
100
R² = 0.984
% drug released
80
60
40
20
0
0 0.5 1 1.5 2 2.5 3
Sq. root of time
c. Square root of time Vs % drug released

Higuchi diffusion mechanism
Figure 3.28 In vitro drug release kinetics of GS: Gelatin: Na alginate microspheres [Formulation: GAM9.1]
Table 3.24 Regression parameters of different release kinetic models

Sr. No. Release Kinetic Model Regression equation R2 t50%
1 Zero order Y =11.58x + 11.57 0.968
2 First order Y = -0.195x + 2.138 0.892 3.55 hrs
3 Higuchi Y = 35.22x + 5.190 0.984
120
y = 7.766x + 11.60
100
R² = 0.965
% drug released
80
60
40
20
0
0 5 Time (hrs) 10 15
a. Percent lidocaine HCl release Vs time (hrs)

Zero order release
2.5
2 y = -0.147x + 2.136
R² = 0.887
1.5
0.5
0
0 5 Time (hrs) 10 15

First order release
120
y = 29.01x - 5.173
100
R² = 0.988
% drug released
80
60
40
20
0
0 1 2 3 4
Sq. root of time
c. Square root of time Vs % drug released

Higuchi diffusion mechanism
Figure 3.29 In vitro drug release kinetics of LH: gelatin: Na alginate microspheres [Formulation: LAM9.1]
Table 3.25 Regression parameters of different release kinetic models

1 Zero order Y = 7.766x + 11.60 0.965
3 Higuchi Y = 29.01x – 5.173 0.9772
In vitro release behaviour of formulation GAM9.1 of GS microspheres was found to

sustain the drug release for 8hrs exhibiting zero order release (R2= 0.968) with Higuchian
diffusion mechanism (R2=0.984) and t50% value of 3.55hrs [Figure 3.28; table 3.24].
Whereas, formulation LAM9.1 of LH microspheres was found to sustain the drug release
for 12hrs exhibiting zero order release (R2= 0.965) with Higuchian diffusion mechanism
(R2=0.9772) and t50% of 4.72hrs [Figure 3.29; table 3.25]. Sodium alginate was found to
extend the release of GS and LH from microspheres.
Residual solvents by Gas Chromatography

The developed microspheres are incorporated into the sponge for sustained drug delivery
so it should be analysed for residual solvent content to avoid toxic effects of solvents.
Formulation GAM9.1 and LAM9.1 were analyzed for residual solvents by method
described in section 3B.3.2.
The results of gas chromatographic studies are given in figure 3.30; tables 3.26 and 3.27.
Figure 3.30 Gas Chromatogram of standards – Acetone and Isopropyl alcohol
Table 3.26 Peak areas obtained for standard solvents mixture and microsphere samples
Sr. No. Peak Area

Standards Solvents GS Microsphere Sample LH Microsphere Sample
Acetone IPA IPA Acetone Fig.5D:IPA
Drug loaded microspheres
Acetone
Sample 1 3355970 1153032 4873 1584 3072 1420
Sample 2 3544644 1113163 5056 1602 3058 1510
Sample 3 3664949 994260 4884 1605 3182 1498
Average 3521854 1086818 4937 1597 3104 1476
SD± 155745.09 82599 102.63 11.36 67.91 48.87
% RSD 3.61 6.21 6.63 1.31 6.63 4.25
Table 3.27 Concentration of residual solvents and pharmacopoeial limits

GS Microsphere Sample LH Microsphere Sample
Particulars IPA Acetone IPA Acetone
Standard peak area observed 1086818 3521854 1086818 3521854
Concentration in µg/ml 50 50 50 50
Sample peak area observed 4937 1597 3104 1476
Sample concentration (µg/ml) 0.2271 0.02267 0.1428 0.02095
Concentration (µg/100mg) 2.271 0.2267 1.628 0.2095
Acceptable Limits given in USP 5000 µg/ml
In the estimation of residual solvents by gas chromatography, the peaks of acetone and
isopropyl alcohol were observed at 2.758 and 2.944 min respectively [Figure 3.30]. The
concentrations of residual solvent in the microspheres were calculated using the peak
areas obtained from standard solvent mixtures. AUC were found to be 0.2271 and
0.02267µg/ml of microspheres for acetone and isopropyl alcohol for GS microspheres
whereas 0.1428 and 0.02095µg/ml of microspheres for acetone and isopropyl alcohol
respectively for LH microspheres [Table 3.27].
The results revealed that the levels of residual solvents acetone and isopropyl alcohol in
final microspheres formulations were well below the acceptable limits given in USP NF
30, limits of 5000μg/ml. The results confirmed the acceptability of the developed
microspheres for incorporation into the biodegradable haemostatic sponges.
Characterization and evaluation of formulation GAM9.1 of GS: gelatin: sodium alginate

microspheres and LAM9.1 of LH: gelatin: sodium alginate microspheres are reported in
table 3.28a and 3.28b respectively.
Table 3.28a Results of evaluation of GS: gelatin: sodium alginate microspheres

[Formulation GAM9.1]
1 Physical appearance and surface Free flowing, pale yellow coloured, individual,
2 Yield %w/w 90± 0.8
4 Mean particle size (µm) 90.52± 2.4
9 In vitro release profile Zero order release within 8hrs
10 Residual solvents Less than 5000 µg/ml
Table 3.28b Results of evaluation of LH: gelatin: sodium alginate microspheres

[Formulation LAM9.1]
1 Physical appearance and surface Free flowing, off white coloured, individual,
2 Yield %w/w 91± 1.2
4 Mean particle size (µm) 110.32 ± 3.4
9 In vitro release profile Zero order release within 12hrs
10 Residual solvents Less than 5000 µg/ml
Optimized formulations GAM9.1 and LAM9.1 of GS and LH microspheres were utilized

further for development of drug loaded gelatin sponges as described in section 3B.4.
3B.4 Incorporation of microspheres into biodegradable sponges

Absorbable gelatin sponges used as haemostatics and coagulants in surgical procedures
can also be used as drug carriers. It can be utilized for development of drug loaded
gelatin sponges. Although gelatin sponges have high elasticity and can be well hydrated
because of their high porosity and large surface area, the drugs implanted in or onto
sponge lamella can only be retarded for a short period of time [Simamora PY, et.al;
1996]. For sustained drug delivery from gelatin sponges, the drug should be incorporated
using sustained release microspheres.
Optimized formulations GAM9.1 and LAM9.1 of GS and LH microspheres as described
in section 3B.3 were used for formulation of GS and LH loaded gelatin sponges
respectively. GS and LH microspheres were incorporated into gelatin sponges separately
and analysed respectively by following methods.
3.4.1 Experimental
Accurately weighed of microspheres (equivalent to 100mg of drug) were incorporated
into gelatin mixture with varying concentrations of 4, 5 and 6% of gelatin in 100ml
o
distilled water. The mixture was generated into stable foam at 40 C with stirring rate
1400-1500rpm. Stable foams were then crosslinked with formaldehyde solution with
varying concentrations of 0.02, 0.04 and 0.06% w/w and characterized for foam
properties as described in section 3A.1.3. Compositions were optimized for crosslinking
degree and whipping duration as described in section 3A.3.1. The composition of drug
loaded gelatin sponge and resulting foam properties and crosslinking degree are given in
table 3.29a for GS loaded sponges and 3.29b for LH loaded sponges.
o
The crosslinked foam was poured into trays and freeze dried at -35 C, under a vacuum of
0.01mbar for 16hrs as per optimized Lyophilization cycle as described in section 3A.2.
GS microspheres of formulation batch GAM9.1 and LH microspheres of formulation

batch LAM9.1 was used for preparation of drug loaded sponges. Weight of 100mg of GS
and LH microspheres were used for 100ml gelatin solution for development of GS and
LH loaded sustained release sponges respectively.
Table 3.29a Composition of GS microsphere loaded gelatin sponges

Formulation Gelatin Formaldehyde Crosslinking Foam properties
code (%) (%w/w) degree (%) 15 min 30 min 45 min
GLS1 0.02 - Unstable Unstable Unstable
GLS1.1 4 0.04 - Unstable Unstable Unstable
GLS1.2 0.06 - Unstable Unstable Unstable
GLS1 0.02 8 Stable Stable Stable
GLS1.1 5 0.04 14 Stable Stable Stable
GLS1.2 0.06 24 Stable Stable Stable
GLS1 0.02 6 Stable Stable Stable
GLS1.1 6 0.04 10 Stable Stable Stable
GLS1.2 0.06 15 Stable Stable Stable
Table 3.29b Composition of LH microsphere loaded gelatin sponge

Formulation Gelatin Formaldehyde Crosslinking Foam properties
code (%) (%w/w) degree (%) 15 min 30 min 45 min
LDS1 0.02 - Unstable Unstable Unstable
LDS1.1 4 0.04 - Unstable Unstable Unstable
LDS1.2 0.06 - Unstable Unstable Unstable
LDS1 0.02 12 Stable Stable Stable
LDS1.1 5 0.04 25 Stable Stable Stable
LDS1.2 0.06 32 Stable Stable Stable
LDS1 0.02 10 Stable Stable Stable
LDS1.1 6 0.04 17 Stable Stable Stable
LDS1.2 0.06 20 Stable Stable Stable
For GS loaded sponge, formulation GLS1.2 was found to give stable foam and
crosslinked sponge with optimum crosslinking degree of 24% and residual formaldehyde
less than 0.001% on whipping duration of 15 minutes.
For LH loaded sponge, formulation LDS1.1 was found to give stable foam and
crosslinked sponge with optimum crosslinking degree of 25% and residual formaldehyde
less than 0.001% on whipping duration of 15 minutes.
3.4.3 Characterization of drug loaded sponges

Developed GS and LH loaded sponges were characterized for appearance and physical
characteristics, morphology, moisture content, water absorption, residual formaldehyde
content as per the methods described under section 3A.3.1.
Determination of drug content

o
The developed sponges of 5x5x1cm size were soaked overnight at 25 C in 100ml of
phosphate buffer, pH 7.4. After filtration through a cellulose acetate membrane (0.65µm),
the concentration of drug in the solution was determined by developed method of
analysis as described in section 2B. The concentration of GS and LH per cm3 was
determined.
In vitro drug release from sponges

This study was performed using USP dissolution rate tester apparatus I. Sponges of
1cm3was placed in basket which was then dipped in a 100mL phosphate buffer pH 7.4
contained in 900ml vessel of the USP dissolution rate test apparatus. The drug release
o
study was performed at 37 ± 0.5 C and the basket was rotated at a speed of 50rpm. Five
ml samples were withdrawn after 0.25, 0.5, 1, 2, 4, 8 and 12 hrs, filtered through
cellulose acetate membrane (0.65μm) and replaced with fresh medium. The drug in the
solution was determined by the method mentioned in chapter 2B, for respective drugs.
All the release experiments were run in triplicate. The release data obtained was
subjected to kinetic treatment according to zero, first, and Higuchi diffusion models. The
correlation coefficient (r), the order of release pattern, and t50% values were determined in
each case.
Dissolution medium uptake

A sponge was accurately weighed and placed in a small bottle containing 30ml of
o
phosphate buffer (pH 7.4) at 25 C. The bottle was turned up and down twice to ensure
complete wetting of the sponge. The sponge was removed from the buffer solution after
15, 35, and 45 minutes by means of small forceps, allowed to drain by careful dropping
on a filter paper, and reweighed. The increase in weight represents the weight of the
buffer solution taken by the sponge, which was calculated as a ratio of the weight of
absorbed buffer solution to the weight of the dry sponge at each period of time as
follows:
Dissolution medium uptake capacity (g/g) = (Wwet-Wdry)/Wdry
This process was repeated till the constant weight of sponges was achieved.
In vitro biodegradation behaviour

In vitro biodegradation of GS and LH loaded sponges was studied by following the
method described in section 3A.3.3. The results are shown in figure 3.33a and 3.33b.

Development of microspheres loaded biodegradable sponges
GS and LH loaded sponges were prepared by incorporating respective
microspheres during the step of mixing the gelatin and formaldehyde before
foaming of the gelatin solution. Since plain gelatin sponges were prepared using
5% of gelatin, gelatin concentration was varied from low 4% and high 6% of
gelatin. Similarly, formaldehyde concentration was varied from 0.02 to
0.06%w/w [Table 3.29a; 3.29b].
The resulting foams were characterized for foam properties by altering whipping
o
duration, freeze dried at -35 C, under a vacuum of 0.001mbar for 12hrs and
evaluated for crosslinking degree.
Optimization of method
Formulation GLS1.2 with 5% gelatin concentration, 0.06%w/w formaldehyde and
15 minutes of whipping time at 1400-1500 rpm resulted in stable foam with 24%
of crosslinking degree for GS loaded sponges.
Formulation LDS1.1 with 5% gelatin concentration, 0.04%w/w formaldehyde and
15 minutes of whipping at 1400-1500 rpm resulted in stable foam having 25% of
crosslinking degree for LH loaded sponges.
Optimum crosslinking degree was achieved with 0.06% and 0.04% wt of
formaldehyde for GS and LH loaded sponges respectively. This can be due to
increase in amino groups in the gelatin solution for which more amount of
formaldehyde is required for crosslinking.
Morphology of microsphere loaded sponges
Photomicrographs for GS and LH loaded sponges are shown in Figures 3.31a and
3.31b. SEM images of crosslinked GS and LH loaded sponges revealed presence
of microspheres on the lamellae and within the pores of the sponges [Figures
3.31B; 3.31C]. This would provide a large surface area for release of drug from
the microspheres.
GS loaded sponges were pale yellow while LH loaded sponges were off white,
both with pore size (80-120μm), wall thickness (3-5μm) with visible microspheres
in the matrix. Both indicated 45-50 times water absorbing capacity. The residual
formaldehyde content was less than 0.001% indicating safe use for wound
application.
A] Photomicrographs [Drug loaded microspheres are demoted by white arrows]
a. GS loaded sponges b. LH loaded sponges

B] SEM images of GS loaded sponge lamellae
a. Microspheres in GS loaded sponge b. Microspheres in LH loaded sponge

C] SEM images of LH loaded sponge
a. Microspheres in GS loaded sponge b. Microspheres in LH loaded sponge

Figure 3.31 Morphology of sponges
In vitro drug release from sponges

This study was performed using USP dissolution rate tester apparatus I using phosphate
buffer, pH7.4 as dissolution medium.
The release data obtained was subjected to kinetic treatment according to zero, first, and
Higuchi diffusion models. The correlation coefficient (r), the order of release pattern, and
t50% values were determined in each case.
Figure 3.32A In vitro drug release kinetics of GS loaded sponges

120
y = 14.55x + 17.41
100
R² = 0.904
% drug released
80
60
40
20
0 2 4 6 8
Time (hrs)
a. Percent drug released Vs time (hrs)

2.5
y = -0.204x + 2.008
2 R² = 0.979
1.5
0.5
0 2 4 6 8
Time (hrs)

120
y = 39.78x - 0.033
100
R² = 0.995
% drug released
80
60
40
20
0 1 2 3
Sq. root of time
c. Percent drug release Vs square root of time

Table 3.29A Regression parameters of different release kinetic models
1 Zero order Y = 14.55x + 17.41 0.9606
3 Higuchi Y = 39.78x -0.033 0.995
Figure 3.32B In-vitro drug release kinetics of LH loaded sponges

120
y = 10.49x + 20.22
100
R² = 0.863
% drug released
80
60
40
20
0 2 4 6 8 10
Time (hrs)
a. Percent drug released Vs time (hrs)

2.5
y = -0.166x + 1.996
R² = 0.976
2
1.5
0.5
0 2 4 6 8 10
Time (hrs)

120
y = 33.89x + 2.419
100 R² = 0.992
% drug released
80
60
40
20
0 1 2 3
Sq. root of time
c. Percent drug release Vs square root of time
Table 3.29B Regression parameters of different release kinetic models

1 Zero order Y =10.49x + 20.22 0.863
2 First order Y = -0.166x + 1.996 0.9715 4.17hrs
3 Higuchi Y = 33.89x + 2.419 0.992
In-vitro drug release profiles of GS and LH from sponges are illustrated in figures
3.32A and 3.32B. Both the formulations showed sustained release of GS and LH
for 6 and 8hrs. This is thought to be attributed to the formation of a dense matrix
as observed in SEM images [Figure 3.31C] which is characterized by smaller pore
sized structures of microspheres and reduced permeability to either dissolved drug
or dissolution medium.
Regression analysis of kinetics of dissolution curve showed that t50% of GS from
microspheres loaded sponges was decreased to 3.39 hrs from 3.55 hrs of GS
microspheres. The t50% of LH from microspheres loaded sponges was decreased to
4.17 hrs from 4.72 hrs of GS microspheres, which was not much significant. Little
difference in t50% might be due to loss of some amount of drug during
incorporation of microspheres into gelatin solution.
It was observed from dissolution kinetics of GS loaded sponges [table 3.30] that
R2 value of first order (0.979) was higher than zero order (0.9606) and R2 value of
Higuchi release kinetics was found to be 0.995 which indicates first order drug
release by Higuchian diffusion mechanism. Same was the case with LH release
from LH loaded sponge which has R2 values of first order (0.9715) higher than
zero order (0.9377) whereas, R2 value of Higuchi release kinetics was observed to
be 0.9905 which indicates first order drug release by Higuchian diffusion
mechanism.
Dissolution medium uptake

Dissolution medium uptake rate of plain gelatin sponges was very fast as it absorbed
dissolution medium within 10 minutes. Indeed, the addition of GS and LH microspheres
significantly decreased rate of water uptake of gelatin sponges to 35 and 40 minutes.
Drug recovered from GS and LH loaded sponges was almost 97±0.6% and 96±0.6% of
GS and LH.
In-vitro biodegradation behaviour

In vitro biodegradation behaviour of drug loaded sponges was carried out using pepsin as
proteolytic enzyme as described in section
Biodegradation behaviour of GS and LH loaded sponges is shown in figures 3.33.
120
Partially degraded soft
gelatinous mass of
100
microspheres remnants
Weight remaining (%) degraded after 80hrs
80
60
40
20
0
0 20 40 60 80
Tim e (hrs)
a. GS loaded sponge
120
Partially degraded soft
100 gelatinous mass of
microspheres remnants
80 degraded after 80hrs
60
40
20
0
0 20 40 60 80
Tim e (hrs)
b. LH loaded sponge
Figure 3.33 In vitro biodegradation behaviour of drug loaded sponges
Error bar indicated standard deviation
GS and LH loaded sponges were found to biodegrade in vitro within 72 hrs leaving soft
gelatinous mass and remnants of microspheres in biodegradation medium which was
found to degrade completely within 80 hrs. Partially degraded soft gelatinous mass of
gelatin was analysed for GS and LH drug content as described in section 2B. It did not
show presence of drug.
Characterization of drug loaded gelatin sponges are summarized in table 3.30.
Table 3.30 Characterization and evaluation of drug loaded gelatin sponges

Parameter GS loaded sponge LH loaded sponge
Appearance Pale yellow, porous sponge with Off white, porous sponge with visible
visible microspheres in the matrix. microspheres in the matrix.
Solubility Insoluble but swells in water. Insoluble but swells in water.
Loss on drying 10% 8%
Digestibility 75 min 80 min
Dissolution medium 40-50 40-50
uptake (g/g)
Formaldehyde <0.001% <0.001%
Pore size 80-120um 80-120um
Wall thickness 3-5um 3-5um
% drug content 97 ± 0.2 96 ± 0.6
t50% (hr) 3.39 4.17
Conclusion
GS and LH loaded microspheres prepared using gelatin and sodium alginate in
combination were successfully incorporated into gelatin sponges. Sustained release from
drug loaded sponges was observed for 6hrs with GS and for 8hrs with LH. Investigation
of characteristics of drug loaded gelatin sponges revealed their potential to be used as
carriers for antiseptic and anaesthetic drugs in addition to haemostatic action.

Research Project On Gelatin

Încărcat de

Informații document

Titlu original

Drepturi de autor

Formate disponibile

Partajați acest document

Partajați sau inserați document

Opțiuni de partajare

Vi se pare util acest document?

Este necorespunzător acest conținut?

Drepturi de autor:

Formate disponibile

Research Project On Gelatin

Încărcat de

Drepturi de autor:

Formate disponibile

Chapter 3A Formulation and evaluation of absorbable gelatin sponges

Biodegradable haemostats [Absorbable gelatin sponges] and Hydrogels are a part of

3A Development of absorbable gelatin sponges as biodegradable haemostat

Procedure for formulation of such therapeutic sponges is described in US patent of 1949.

Design and development of surgical dressings for advanced wound management 91

Gelatin, a natural protein is used in biodegradable materials. It is converted into other

Design and development of surgical dressings for advanced wound management 92

Reaction of formaldehyde with gelatin

Figure 3.1 Mechanism of crosslinking with formaldehyde

Design and development of surgical dressings for advanced wound management 93

Gelatin foam was optimized and characterized for following parameters

3A.1.2 Optimization of methods

Design and development of surgical dressings for advanced wound management 94

Optimization of concentration of gelatin solution

Optimization of pore size and wall thickness

Optimization of whipping condition

Design and development of surgical dressings for advanced wound management 95

Figure 3.2 Measurement of foam density and surface characteristics

Formulation Gelatin solution

Gelatin concentrations of 4% and 5% were found to give stable foam but 5%

Design and development of surgical dressings for advanced wound management 96

Design and development of surgical dressings for advanced wound management 97

Formulation Gelatin solution Concentration of Wall thickness

GS5.1 0.1 Unstable 80-150 2.5-3.5

Design and development of surgical dressings for advanced wound management 98

Table 3.3 Effect of speed of agitation of stirrer on pore size of sponges

Speed of stirrer. Foam volume as

Table 3.4 Effect of whipping duration on Gelatin foam properties

3A.1.3 Results and Discussion

Design and development of surgical dressings for advanced wound management 99

3A.2 Drying of absorbable gelatin sponges by Lyophilization

Basic Understanding of Freeze-Drying Equipment and process

Figure 3.4 Lyophilizer: LYOVAC [Laboratory Scale]

3A.2.1 Development and Optimization of lyophilization cycle

Figure 3.5 DSC Thermogram of porcine gelatin

Table 3.5 Optimization of lyophilization cycle

Duration of Moisture content of

3A.2.2 Results and discussion

3A.3 Characterization and evaluation of plain gelatin sponges

3A.3.1 In-vitro characterization

Appearance and physical characteristics

ii. Wall thickness

Residual formaldehyde content

Measurement of degree of Crosslinking

Absorbance of crosslinked sponge

After determination of degree of crosslinking, residual formaldehyde content for each

In-vitro biodegradation of gelatin sponges

Random coiled polypeptides

Amino acids & Short peptides

Accordingly, experiment was designed to check the in vitro biodegradation of gelatin

Sterilization of gelatin sponges

Sterilization is an important consideration in the design of surgical dressings which are

Prepared Gelatin Sponge Packing of prepared gelatin sponge

Comparison with marketed gelatin sponges

3A.3.2 Results and discussion

A Developed gelatin sponge

Figure 3.7 Comparative morphology of gelatin sponges

FT-IR spectroscopy for determination of crosslinking

Figure 3.8 FT-IR spectra of gelatin and crosslinked gelatin sponge

A. FTIR spectra of crosslinked uncrosslinked gelatin where peak assignment is as follow:

B. FTIR spectra of crosslinked gelatin sponge where peak assignment is as follow:

FT-IR spectroscopic analysis of uncrosslinked gelatin and crosslinked gelatin sponges

Measurement of degree of Crosslinking