In: Gelatin: Production, Applications and Health Implications ISBN 978-1-62417-627-2

Editor: Gökhan Boran, pp. 49-70 © 2013 Nova Science Publishers, Inc.

Chapter3

GELATIN BASED MATRICES FOR DRUG DELIVERY

APPLICATIONS

Goutam Thakur1, Dérick Rousseau2*, Ruby Rose Rafanan2

1
Department of Biomedical Engineering, Manipal Institute of Technology, Manipal
University, Manipal-576104, Karnataka, India
2
Department of Chemistry and Biology, Ryerson University, Toronto, Canada, M5B 2K3

Abstract
This chapter highlights select gelatin-based carrier matrices which are seeing more frequent
use as drug delivery systems. Gelatin gels, alone and in combination with other polymers, can
act as carriers for the delivery of various bioactive molecules. Controlled release systems and
their underlying mechanisms along with mathematical modeling are outlined to show the
various means by which target compounds are released from gelatin-based systems.
Biocompatibility and applications of gelatin-based carriers in the fields of medicine and
biomedical research are discussed.

Keywords: Gelatin, carrier matrix, drug kinetics, controlled release, biocompatibility

Introduction
Controlled release systems for drugs and bioactive agents help to optimize their therapeutic
benefits by ensuring their efficient delivery to targets sites. Conventional drug delivery
methods viz., injections, tablets and sprays etc., used for administering bioactive molecules
often require repetitive and frequent administration to achieve adequate therapeutic benefit.
Systemic drug concentrations may fluctuate due to inconsistent dosing stemming from
improperly-regulated administration time and dose concentration. In some cases, the drug
may accumulate within the body, exceeding the therapeutic upper limit, inducing adverse
effects. Alternatively, long gaps between doses may cause bodily concentrations to fall below
the minimum effective value, rendering the drug essentially ineffective (Figure 1).

*
E-mail address: rousseau@ryerson.ca
 Thakur et al.

Pharmaceuticals with complex dosing patterns clearly benefit from controlled release systems
that optimize drug pharmacokinetics and pharmacodynamics [1]. Present day controlled
release systems may consist of natural or synthetic polymeric carriers (e.g., capsules, gels,
beads, etc.) through which a drug can be delivered to its target area [2, 3, 4]. Advantages to
these systems include the maintenance of drug levels within a desired therapeutic range, the
need for fewer administrations, and increased consumer compliance. However, potential
disadvantages such as the possible toxicity or bio-incompatibility of the materials used,
undesirable by-products of degradation, surgical intervention related to implantation devices
and possible discomfort caused by the device cannot be ignored. An ideal drug delivery
system should be inert, biocompatible, mechanically strong, and comfortable for the patient,
capable of achieving high drug loading, safe from accidental release; and simple to
administer, fabricate and sterilize.

Figure 1: Plasma drug levels following administration of a drug from a conventional standard
dosage form (a), overdose (b), and controlled release dosage form (c). The maximum and
minimum therapeutic levels are represented by (- - -) (reproduced with permission from [5]).

Physical configuration of gelatin-based controlled release matrices

Gelatin-based matrices have been developed in various shapes and categorized into different
delivery systems (viz., hydrogels, sponges, microparticles, microspheres, sheets, emulsion
gels, nano-carriers, etc.) for the delivery of drugs by oral, transdermal, ophthalmic or
 Gelatin Based Matrices For Drug Delivery Application 61

parenteral routes [6-10]. With the advent of nanotechnology, there has been significant
progress in developing nano-sized drug delivery systems, whose typical size varies from 1 nm
to 100 nm. The bioactive agents may be uniformly distributed within nano-carriers (e.g.,
nanoparticles, nanosphere, nanorods or nanocapsules) or may be adsorbed onto their surface
[11]. Some carriers may be designed in a way that both hydrophobic and hydrophilic drugs
can be accommodated within their structure [10]. This versatility in forming various
structures with different physical (and chemical) characteristics is the primary reason why
gelatin-based systems can be adapted to serve various purposes, especially as a controlled
release matrix.

Controlled release mechanisms

There are three primary mechanisms by which active agents can be released from a delivery
system: diffusion, degradation, and swelling followed by diffusion [12]. Any or all of these
mechanisms may occur in a given release system. Release mechanisms can be chemical or
physical in nature and usually involve diffusion – the movement of individual drug molecules
or other active agents occur through a semi-permeable barrier from an area of higher
concentration to an area of lower concentration. Diffusion can occur through pores in the
polymer matrix or at a molecular level, by passing through the interstitial space between
polymer chains or following degradation of the carrier matrix. Movement of the active agent
is primarily reliant on the properties of the polymeric network, solvent–polymer interactions,
and varies in magnitude depending on the phase; it is fastest in gases (~10 cm min–1), slower
in liquids (~0.05 cm min–1) and slowest in solids (~0.00001 cm min–1). Diffusion in hydrogels
is more complex and the diffusivities of incorporated compounds will lie somewhere between
those observed in liquids and solids. Physically, diffusion-controlled release is classified into
two types, depending on its mode of release: (a) reservoir-type, or (b) monolithic diffusion,
the latter is also known as matrix-type diffusion [5, 13, 14] (Figure 2).

Reservoir-type

In this type of release matrix, therapeutic compounds (solid or liquid) form a core surrounded
by a microporous or non-porous polymeric network (e.g., membranes, hydrogels,
microcapsules, liposomes, hollow fibres, etc.) [5]. If the therapeutic agent is saturated, its
transport will be constant and it will follow zero-order release kinetics since its chemical
potential will remain unaffected [14-16]. However, even under ideal conditions, release is
generally not constant during the initial and final release phases [14, 15]. When immersed in a
releasing medium, the system will take time to attain its steady state. In the presence of little
or no therapeutic compound near the surface, an induction period will be required for
saturation of the surface by the therapeutic agent. In contrast, if the therapeutic compound is
concentrated near the surface, the initial release rate will be higher than the equilibrium-state
value due to an initial ‘bursting’ effect. As release nears completion, the therapeutic
compound concentration within the core will drop and the release rate will decrease. In such
systems, drug diffusion through a polymer matrix is a rate-limiting step and the release rate is
reliant on the choice of polymer and its effect on both diffusion and the partition coefficient
of the drug molecules to be released [5].
 Thakur et al.

Matrix-type

In this scenario, the therapeutic agent is dissolved or dispersed within a polymer network.
Release is governed by various parameters, namely solubility of the therapeutic compound, its
diffusivity in the polymer phase and polymer–compound interactions. As well, shape and path
length also play crucial roles [14, 17]. Release occurs due to diffusion of the drug throughout
the macromolecular mesh or solvent-filled pores. The fractional release from a one-
dimensional device can be modeled using Fick’s 2nd law. In these systems, the release rate is
proportional to time to the one-half power, rendering time-independent or zero-order release
in this type of system with simple geometries impossible. Drugs can be incorporated into
gelatin-containing gels by equilibrium partitioning, where the gel is swollen to equilibrium in
a concentrated drug solution/dispersion; or during a polymerization reaction. Equilibrium
partitioning is a more favorable loading method for drug-polymer systems with large partition
coefficients or for sensitive macromolecular drugs such as peptides or proteins that may
degrade during polymerization.

Swelling-controlled release systems

In such systems, the drug is dispersed within a glassy polymer that prevents drug diffusion
until there is contact with biological fluid, at which point the polymer begins to swell. The
penetrant fluid enters the glassy polymer, causing a reduction in the glass transition
temperature of the polymer that allows for local relaxation of the macromolecular chains.
More specifically, the presence of solvent in the glassy polymer causes stresses which are
then accommodated by an increase in the radius of gyration and end-to-end distance of the
polymer molecules, i.e., the polymer chains become solvated. The drug is then able to diffuse
out of the swollen, rubbery area of the polymers presuming the mesh size between the matrix
polymer chains is sufficiently large to permit passage. This type of system is characterized by
two moving fronts: i) the front separating the swollen (rubbery) portion and the glassy regions
which moves with a certain velocity and ii) the front located at the polymer-fluid interface.
Generally, solvent molecules move simultaneously into the glassy polymer matrix with a
well-defined front at a particular velocity. The time taken for the increase in the radius of
gyration of the polymer molecules is characteristic for a particular polymer/solvent system
[18].

Biodegradable controlled release system

With the previously-mentioned systems, polymers only undergo physical changes. In

contrast, biodegradable polymer-based delivery vehicles undergo chemical degradation
releasing active agents into the medium. Most biodegradable polymers are designed to
degrade into small, biologically acceptable fragments. In such systems, the drug is uniformly
distributed throughout the polymer carrier. As the polymer surrounding the drug erodes, it is
released into the surrounding environment. The main advantage of using this delivery system
is that it degrades within the body as a result of natural biological processes, thus eliminating
the need to withdraw the system after completion of release. Ideally, the drug is released only
as a result of surface erosion of the polymer. In such systems, there are three major
mechanisms by which the polymer degrades: Type 1, where water-soluble polymers erode
 Gelatin Based Matrices For Drug Delivery Application 63

due to hydrolysis of water-labile linkages, enzymatic degradation, or simple dissolution of

physical crosslinks; Type 2 erosion occurs via solubilization of insoluble or hydrophobic
polymers and occurs as a result of hydrolysis or ionization, and Type 3 deterioration occurs as
a result of polymers fragmenting into small, water-soluble portions [5].

Figure 2: Drug release from (A) reservoir-type (B) matrix-type (C) swelling-controlled and
(D) biodegradable drug delivery system (reproduced in modified form with permission) [19].

Release kinetics and mathematical modeling

Several models describe diffusion/dissolution from immediate and modified release dosage
forms. Experimental data is correlated with a theoretical model to better elucidate the actual
mechanism of release. Many parameters such as particle size and solubility are reported to
influence release kinetics. Taking the desired administration method into consideration, as
well as dose and targeted drug release profile, mathematical predictions allow for accurate
estimates of the required composition, geometry, dimensions and preparation procedure of the
final product. The underlying drug release mechanisms can be elucidated by systemic
quantitative analysis of the physical, chemical and potentially biological phenomena which
are involved in the control of drug release. Most theoretical models are based on diffusion and
are intimately connected with structure and morphology of the material through which the
diffusion takes place [20]. Fick’s law of diffusion is often used to describe release [20].

(1)
 Thakur et al.

(2)

where the concentration and mass flux of species i are designated as ci and ji, respectively; Dip
represents the diffusion coefficient of i in a polymer matrix and x and t are independent
variables for position and time, respectively. Although the diffusion coefficient is assumed to
be independent of concentration, this model incorporates concentration into the diffusion
coefficient to maintain computational clarity [20]. To correct for these assumptions, a
concentration-dependent diffusion coefficient, ( ) is used in equations (1) and (2); z is
the thickness of matrix. After establishing initial and boundary conditions, Equation 2 is
expressed with the under the defined conditions and rewritten as:

( ( ) ) (3)

Here, ( ) is influenced by the structural characteristics of the polymer carrier.

Eyring’s theory [21] was one of the earliest known descriptions of the diffusion coefficient
through a polymer carrier. According to this theory, the diffusion of solute through a medium
is presented as a series of discrete, incremental increases of a certain frequency rather than a
continuous process.

(4)

Equation 4 is derived from the Eyring theory [21] where is the diffusional increase of the
drug in the polymer and is the frequency of this change.

Fujita [22] introduced the idea of free volume in polymers for estimation of the drug diffusion
coefficient. The exponential dependence of the drug diffusion coefficient on the free volume
is expressed in Equation 5 and was later refined by Yasuda and Lemase [23] (Equation 6).

{ } (5)

( ) [ ( )( )] (6)

Here, the normalized diffusion coefficient is the ratio of the diffusion coefficient of the solute
in the polymer, , to the diffusion coefficient of the solute in the pure solvent, . This
ratio is related to the degree of hydration H and free volume is occupied by the swelling
medium . The sieving factor excludes drugs of a cross-sectional area of . B is a
parametric characteristic of the polymer. The subscripts 1, 2 and 3 in Equation 6 represent the
swelling medium, drug and polymer, respectively.

Peppas and Reinhart [24] developed a theoretical model based on the free volume theory of
the polymer matrix expressed as:
 Gelatin Based Matrices For Drug Delivery Application 65

̅ ̅
(̅ ̅
) ( ) (7)

where the normalized diffusion coefficient is related to the degree of swelling Q, the solute
radius rs and the molecular weight of the polymer chains. The polymer chain network is in
part described in terms of molecular weight. These parameters are defined as ̅ , the average
molecular weight of polymer chains between adjacent crosslinks, ̅ , the average molecular
weight of the linear polymer chain without crosslinking and ̅ , the critical molecular weight
between crosslinks, below which a drug of size rs, cannot diffuse through the polymer
network. Constants and are associated with the polymer structure. This theory is
limited to drug delivery in highly swollen, nonporous hydrogels [20].

Kinetic models

There are a number of kinetic models that describe the overall release of drugs from various
dosage forms. Qualitative and quantitative changes in a formulation may alter drug release
and in vivo performance, hence establishing predictive tools that facilitate product
development by ultimately reducing the number of biological studies necessary. Following
are common model-dependent methods.

Zero-order kinetics

Drug dissolution from various dosage forms that do not disintegrate, allowing for slow drug
release can be expressed as:

(8)

where represents the fraction of the drug dissolved at time t and is the apparent
dissolution rate constant or zero-order release constant. Dosage forms following this profile
lead to constant release drug concentrations per unit time and permit prolonged action [25].
The model can be represented in the following way:

(9)

where is the maximum amount of drug released at time t, is the initial amount of the
drug present in the releasing medium ( =0 in most cases) and is the zero-order release
constant.

1st order kinetics

Many researchers have deduced various equations to explain this mode of release, including:

(10)
 Thakur et al.

where is the maximum amount of drug released at time t, is the initial amount of the
drug in the solution and K1 is the first-order release constant. The drug release rate is
proportional to the drug concentration remaining within the interior, in effect, releasing
diminishing quantities of the agent over time.

Higuchi model

This model is applicable to the release of both water-soluble and poorly-soluble drugs
incorporated within a semi-solid or solid matrix. The model assumes that the rate of
dissolution of the active agent is faster than that of its diffusion thus maintaining a continuous
release of the active agent into the surrounding media [26]. The rate equation for this kind of
system has been derived by Higuchi:

(11)

where dM is the change in the amount of drug released per unit area, dx is the change in the
drug-depleted matrix layer thickness; C0 represents total amount of drug per unit volume of
the matrix and Cs is the saturated concentration of the drug in the matrix. Diffusion theory
expresses the rate of mass transfer as per Equations 12 and 13:

(12)

(13)

where Dm is the diffusion coefficient in the matrix. Equating, integrating and solving
Equations 12 and 13 yields:

[ ( ) ]1/2 (14)

When the amount of drug is in excess of the saturation concentration i.e. C0>>Cs, the
equation is simplified as:

[ ]1/2 (15)

or

(16)

where k = CsDm2C0 is constant as the variables Cs, Dm and C0 do not change for a given
delivery system. If a plot of the amount of drug released versus the square root of time is
linear, then the drug release from the matrix is said to be diffusion-controlled and follow
Higuchi [26].
 Gelatin Based Matrices For Drug Delivery Application 67

Weibull model

This model has been described for different dissolution processes as:

( )
[ ] (17)

where M is the amount of drug dissolved as a function of time t and M0 is the total amount of
drug released. T accounts for the lag time measured as a result of the dissolution process; a
denotes a scale parameter that describes the time dependence while b describes the shape of
the dissolution curve progression. For b = 1, the shape of the curve corresponds exactly to the
shape of an exponential profile with the constant k = 1/a (Equation 18).

( )
[ ] (18)

As the value of b decreases, the exponential curve steepens. A b value greater than 1 results
in a sigmoidal plot. The Weibull model is most useful for comparing the release profiles of
matrix-type drug delivery systems [25, 27].

Peppas model

Korsmeyer et al. [28] derived a simple relationship which described drug release from a
polymeric system that has been extensively used.

(19)

where Mt/M is the fraction of drug released at time t, k is the release rate constant and n is
the release exponent. To identify the release mechanism, a known quantity of the drug is
released from a system of a known geometry and fit to the model above. The value of n for a
given geometry designates the release mechanism for the system. The diffusional exponent
values and corresponding release mechanisms are given in Table 1.

Table 1: Diffusional exponent (n) and release mechanism for swellable release systems.
Diffusional exponent, n Release mechanism

Cylindrical Spherical
Thin film sample sample
0.5 0.45 0.43 Fickian diffusion
0.5<n<1.0 0.45<n<0.89 0.43<n<0.85 Anomalous transport
(non-Fickian)
1.0 0.89 0.85 Case II transport
>1.0 - - Super-case II transport
† Source: (Ritger and Peppas, 1987) [29]
 Thakur et al.

Crosslinking

The purpose of crosslinking is to improve the thermal and mechanical stability of the release
matrix under physiological conditions [30]. Crosslinking can also be tailored to modify the
release rate of incorporated active agents by changing the pore size of the crosslinked gelled
matrix [31, 32, 20]. Crosslinking methods are classified into two categories: physical and
chemical. Physical crosslinking is promoted via methods such as irradiation with UV and/or
gamma radiation whereas chemical crosslinking uses various natural and synthetic
crosslinkers like genipin and glutaraldehyde to alter gel strength and release properties [33].
Physical crosslinking often fails to achieve the desired release properties in a matrix as there
may be difficulty in controlling the crosslinking density. Chemical crosslinking agents have
been categorized into two types: non-zero-length crosslinkers and zero-length crosslinkers
[34]. Non-zero length crosslinkers (e.g., glutaraldehyde, polyepoxides and isocyanates) are
bi-functional or multifunctional molecules that operate by bridging free carboxylic acid
groups, amino groups and hydroxyl groups between adjacent polymer molecules. With zero-
length crosslinkers, reactive groups such as carboxylic acid and amine groups present in
polymer network chains react with each other leading to the formation of a covalent bond.
Crosslinking agents in this category include acyl azides, transglutaminase and carbodiimides
[34-36]. Polyphenols (e.g., catechol) have the capacity to form networks with biopolymers in
the presence of catechol oxidase [37]. A brief overview of some crosslinkers of biomedical
importance is highlighted in Table 2.

Evaluation of crosslinking efficacy

Crosslinking density/degree of crosslinking is a useful structural parameter and can be

effectively determined by chemical assays and swelling behavior. In the context of
biopolymer-based gel matrices which contain amino groups, fixatives are added to the
biopolymer solution and chemically interact with available amino groups during the process
of crosslinking. Eventually, this will result in a measurable fraction of amino groups lost in
the process of crosslinking, referred to as the degree of crosslinking.

Yao et al. [38] determined chemical crosslink density using the ninhydrin assay. In their
study, samples were weighed (~20 mg) and subsequently heated with a ninhydrin solution (2
wt % ) at 100 °C for 20 min. Afterwards, the optical absorbance of the solution was recorded
with a spectrophotometer at a wavelength of 570 nm. After heating with ninhydrin, the
number of free amino groups in the test sample was proportional to the optical absorbance of
the solution [39]. Crosslinking index (CI) of the samples were then determined as described
elsewhere [40, 41]. The trinitrobenzenesulfonic acid (TNBS) assay method [42] is
comparable to the ninhydrin assay for determining the number of uncrosslinked amino groups
in a crosslinked matrix. Raman and NMR spectroscopic methods also provide adequate
alternatives in determining the degree of crosslinking [43].
 Gelatin Based Matrices For Drug Delivery Application 69

Table 2: Summary of some crosslinkers used for biomedical applications

Cross linkers Structure Applications References

Formaldehyde Tissue engineering [44-46]

scaffold, stabilizing
matrix

Glutaraldehyde Cell, enzyme [44, 47-52]

immobilization,
hydrogel synthesis, drug
delivery, tissue
engineering

Genipin Stabilizing matrix, tissue [53-56]

engineering, drug
delivery, stem cell
behavior

Crosslinking density from equilibrium swelling and the Flory –Rehner theory

The swelling-dependent crosslinking of polymer gels can be determined using the Flory-
Rehner approach [57]. When a polymeric network swells in a compatible solvent and is
allowed to attain equilibrium, there are only two forces at work – the force of thermodynamic
mixing and the retractile force of the polymer [57, 58]. Crosslinked polymer gels, when
immersed in solvent, swell and eventually attain equilibrium. As a result of solvent ingress
into the polymer network, the volume increases causing the network junction zones to
expand. The swelling reduces the chain configuration entropy. However, there is a concurrent
and opposing increase in entropy during swelling due to mixing of the polymer and solvent.
Equilibrium is reached when these two opposing entropies become equal in magnitude, and
can be expressed in terms of Gibbs free energy:

(20)

where ∆ is the energy change due to the formation of network chains between network
junction zones and ∆ 𝑚𝑖 𝑖 𝑔 is the result of solvent-polymer mixing.
 Thakur et al.

Peppas and Merrill [59] proposed a modified theory that gives some insight into the average
molecular weight of the polymer as well as crosslinking density of a gel prepared in water.
The following equation was derived for the swelling of a hydrogel prepared in the presence of
a solvent [60, 61]:

 V  2M c   v2,s  
1/ 3
 v
2 2
 
V1  v2,s 
 
4 I  v 
K
 pOH b

 
  ln 1  v2, s   v2, s  1v22, s  v2,r  1 1   

   2, s
  2v


 10 K a   v M c  M n   2, r 
v  2, r 
(21) 
where ̅ is the molecular weight of the polymer without crosslinking, ̅ is the number-
average polymer molecular weight between two adjacent crosslinks, ̅ is the specific volume
of the hydrogel prior to swelling, ̅ is the molar volume of the solvent water (18 mL mol-1),
is the polymer volume fraction in the swollen state determined as roughly the inverse of
the equilibrium swelling ratio, is the polymer volume fraction in the relaxed state (the
state of the polymer immediately after crosslinking, but before swelling), is the ionic
strength, and are the dissociation constants for the acidic and basic moieties on the
polymer, and is the Flory-Huggins parameter describing the polymer-solvent interaction
[20, 42]. Using ̅ , the crosslink density, , can be determined using [62]:

Mn
q
Mc (22)

The parameter is determined from the volume-swelling ratio, [63]:

1
 2, s 
qv (23)
The volume-swelling ratio is calculated as [63]:

qv  1 
qw  1   2
1 (24)
where and are the densities of the polymer network and solvent, respectively. The
weight-swelling ratio is determined from [63]:

ms
qw 
mo (25)
where m0 and ms are the mass of the unswollen gel and the mass of the swollen gel at
equilibrium, respectively.

Properties of gelatin

Gelatin is a translucent, colourless powder that is used as a gelling agent in the food industry
given its availability and ease of use. It is a biopolymer of animal origin (i.e., from the skin
 Gelatin Based Matrices For Drug Delivery Application 71

and bones of bovine, porcine and fish sources) derived from the heat-induced hydrolytic
degradation of collagen. It is commonly used in pharmaceutical and (bio)medical applications
given its biodegradability [64-66] and excellent biocompatibility in physiological
environments [38] (e.g., as a plasma expander, an ingredient in drug formulations, and as a
sealant for vascular prostheses) [67, 68]. Either acidic or basic gelatin can be produced
depending on the method of extraction from collagen, with each having a different isoelectric
point (pI) [69]. Alkaline-processed gelatin (which is usually from bovine sources) possesses a
greater proportion of carboxyl groups, rendering it more negatively-charged and lowering its
pI compared to acidic-processed gelatin (usually porcine in origin) which possesses a pI
similar to collagen. Furthermore, depending on type, gelatin can undergo polyion
complexation with either positively or negatively-charged bioactive agents. Acidic gelatin
(pI~5.0) can assist in the controlled delivery and release of basic bioactive agents whereas
basic gelatin (pI ~9.0) is best adapted to acidic compounds [69, 70].

Molecular basis of gelatin sols and gel formation

Gelatin is primarily extracted from type I collagen, whose triple helix structure consists of
two α1 (I) and one α2 (I) chains each having a molecular weight of ~95 kDa [71]. The gelatin
tripeptide is primarily composed of the same three amino acids (glycine, proline and
hydroxyproline: Figure 3A) and is structured similarly to tropocollagen (Figure 3B). The
proline content promotes formation of the polyproline II helix, which determines the form of
tropocollagen, a trimer of ~ 330 kDa [72, 73]. Gelatins with a higher proline and
hydroxyproline content will result in stronger gels having higher gelling temperatures.
Furthermore, glutamic acid, aspartic acid, lysine, arginine and histidine may also form
crosslinks and participate in electrostatic interactions [73-75].

Glycine Proline Hydroxyproline

(A)
 Thakur et al.

O
HN O
N N O OH
H NH
O O
O
N
HN O
N O
O H HN
HN
NH2 N
O
H2N
O

(B)
Figure 3: Chemical structures of the (A) most important amino acids (glycine, proline and
hydroxyproline) of gelatin and (B) structure of gelatin.

The gels formed by gelatin are thermo-reversible in nature. Its gel-to-sol transition takes place
at ~35 oC, i.e., gelatin forms a gel at temperatures below 35oC and has a sol-like consistency
at temperatures above 35oC [76, 77]. The properties above the sol-gel temperature are
attributed to the presence of polypeptide chains which generally exist as isolated, flexible and
lightly crosslinked entities. Upon cooling, the gelatin sol sets as a transparent gel provided
that the concentration is greater than the critical gelling concentration, generally ~0.4-1%.
This is due to a reversion to an ordered triple-helix conformation (physical crosslinking)
along the peptide contour from the disordered coil alignment. At very low concentrations
(<0.1%), intra-molecular bonds are formed preferentially within single chains through
electrostatic interactions. At concentrations ≥ 1%, helix growth induces chain association and
three-dimensional network formation, where two processes are involved: a) the formation of
single helix nuclei and b) the aggregation of these single helices to a triple helix structure.
The extent and manner of reversion to the collagen fold (triple helix) structure is dependent
on solvent, temperature and concentration [78]. Higher gelatin concentrations and slower
rates of cooling allow more inter and intra-molecular crosslink formation [73].

Use of gelatin in carrier matrices

Below are shown various examples of where gelatin has been used for the controlled release
of chemotherapeutic agents and bioactive compounds [8, 79-85]. Prosthetic valve
endocarditis is a serious complication of cardiac valve replacement, in which bacteria that
have entered the site during surgery adhere to the valve and give rise to infection. In an
attempt to reduce the incidence of early infective endocarditis, Kuijpers et al. (2000) [86]
 Gelatin Based Matrices For Drug Delivery Application 73

studied the in vivo and in vitro release of lysozyme from in situ gelatin hydrogels to prevent
valve endocarditis. Other antibiotic delivery applications which have benefited from the use
of gelatin carriers includes the release of tetracycline and bisphosphonate from gelled foam
pellets to reduce periodontal bone loss in a rat model [87] and the release of ciprofloxacin
hydrochloride for the management of ocular infection [88].

Gelatin-based controlled release vehicles specifically designed to deliver chemotherapeutic

agents is an area of significant development, since the therapeutic index of these drugs is
relatively low and clearly benefits from more effective dosing. Localized delivery of a
chemotherapeutic drug to the immediate vicinity of the affected site reduces the total amount
of drug required in the body for effective treatment and also minimizes the severity of side
effects for the patient. In this context, Muvaffak et al. [89] described the use of gelatin
microspheres to deliver colchicine (antimitotic drug) in vitro. These colchicine-loaded
microparticles had a high initial toxic effect on MCF-7 cancer cell lines. Ohta et al. [90]
showed that prolonged cisplatin release from drug-loaded gelatin microspheres and their
improved chemoembolic anti-cancer effect against VX2 liver tumors in rabbits. Overall, these
data suggest that gelatin micro-carriers are potential candidates for mediating sustained
release of chemotherapeutic agents. A diverse range of applications have been studied for
gelatin carrier-mediated drug delivery (Table 3).

Recent advances in the development of gelatin controlled release systems that remain in
circulation may also benefit chemotherapeutic applications as these carriers gradually
accumulate at the tumor site and produce an effect known as “enhanced permeability and
retention” (EPR). For example, by surface-coating a circulating drug carrier with
poly(ethylene glycol) (PEG), uptake by the mononuclear phagocyte system (MPS) after
intravenous injection is minimized, its persistence in the bloodstream is extended, and given
enough time, a chemotherapeutic drug/carrier complex can take advantage of the EPR effect,
essentially achieving a simple form of “drug targeting”. Kushibiki et al. [104] coupled PEG
with an active ester terminal group to the amino terminus of gelatin to prepare PEG-grafted
gelatin. Results indicated that PEG-gelatin with high degrees of PEGylation formed a micelle
structure with its surface consisting of grafted PEG molecules and did not adsorb onto a
gelatin affinity column, in contrast to unmodified gelatin and PEG-gelatin with a low degree
of PEGylation which were both retained on the column. Furthermore, micelle formation by
PEGylated gelatin exhibited a longer residence time within the body than gelatin alone,
making it a potential candidate as a “stealth” drug carrier able to circumvent filtering by the
MPS and thus accumulate at the tumor site via the EPR effect.

Biocompatibility assessment

The use of biomaterials for various biomedical applications requires different in vitro or in
vivo tests to study the safety and efficacy of the materials involved. Biocompatibility refers to
the extent to which the material does not have toxic effects on a biological system. Apart
from being ‘safe’ i.e., biologically compatible with tissues, a biocompatible material should
also maintain its functionality. Figure 4 provides an overview of quantitative analysis in
determining biocompatibility.
 Thakur et al.

Table 3: Selected bioactive agents incorporated in gelatin-based carrier matrices and their
applications
Carrier material Bioactive agent Matrix type Application References
G, PVA/G, GF, BSA , EGF, Hydrogel Controlled release, [70,81,91-97]
Crosslinked G, I-labeled b-FGF, bone regeneration,
β-cyclodextrin/G, IGF-1, lysozyme, corneal epithelial healing ,
erythropoietin, wound healing, ,
simvastatin cartilage tissue engineering
TGF-β1, BMP-2,

G, β-TCP/G Paclitaxel, Sponge Lymphatic metastasis in cancer, [8,98]

BMP-2 Osteoinduction

G Adrenomedullin Microparticle Angiogenesis [99]

G, CMC-G, Ketorolac, Microsphere Controlled delivery, [54,70

G/mucin, G-CAP, Indomethacin, tissue engineering, 100-102]
TGF-β2, tracheal replacement
RITC-Ins,
doxorubicin
ceftrioxone,

G Erythropoietin Sheet Myocardial infraction [7]

G, thiol/G, Suphamethoxazole, Nanoparticle Controlled delivery, [6, 70,103]

PEG/G intracellular DNA gene delivery
plasmid, bFGF,

† G: gelatin; PVA: poly(vinyl) alcohol; GF: growth factor; BSA: bovine serum albumin; EGF: epithelial growth factor;
bFGF: basic fibroblast growth factor; TGF: transforming growth factor; IGF: insulin-like growth factor; RITC-Ins:
rhodamine iso-thiocyanate labeled insulin

To evaluate the biocompatibility of a material, various tests are necessary to execute beyond
simple morphological examination of material-cell interactions. According to Pizzoferato et
al. [104], the individual tests for evaluation of a material’s biocompatibility are not definitive
on their own (Figure 4), however, the results of these tests as a collective help in defining the
biological tolerance of the materials. Preliminary study followed by toxicological
investigation of the material, its function and its changes during use are considered during
biocompatibility assessment. Screening of materials predicts and characterizes potential
toxicity in its various forms. However, to integrate the screening and supplementary tests, it is
necessary to perform assessments on infection-related phenomena and structural testing of
biomaterial or peri-prosthetic tissues. These testing methods are flexible and adaptable, taking
into account the constituent materials and its end use. The scheme further highlights
 Gelatin Based Matrices For Drug Delivery Application 75

evaluation stages involving cell cultures. The first stage includes cytotoxicity tests on cell
cultures. This initial screening aids in selecting suitable materials for implantation as well as
testing. The second stage of testing ensures thorough evaluation of the biocompatibility of the
material in relation to its end use.

Figure 4: Quantitative analysis of biocompatibility (adapted and modified from [105]

Biocompatibility in drug delivery systems

Controlled release systems have been used for many years with immense commercial success.
These developments are especially promising for use in implanted devices. However, when
artificial materials are implanted inside the body for extended periods, adverse responses
become more of a concern. Biomaterials are non-viable materials that remain as part of the
body to restore or replace the normal functions of living systems.

Recently, various drugs have been incorporated into implantable biomaterials to improve
functionality [106]. The development of drug delivery systems is not limited to only the
development of materials suitable to the specific application (biodegradable, pH-sensitive,
anionic, flexible, etc.), optimization of drug loading and type of release kinetics (slow, fast,
pulsatile). Also of chief importance is determining the biocompatibility of a systemically
circulating drug as it functions with its delivery system within the body. Biocompatibility
issues are further complicated by the lack of fully understood drug-tissue interactions and
material properties [107]. Generally, the assessment of biocompatibility progresses through in
vitro and in vivo phases, which in many instances use similar experimental tests. In vitro
evaluation provides a rough assessment of the ability of relevant cell types to survive in the
presence of a material. The MTT assay, measures of DNA synthesis, cell proliferation, and
dye-based cell membrane integrity tests are commonly employed for determining the effects
 Thakur et al.

of both direct contact with cells and indirect exposure to diffusible components. In vivo
studies are also vital in determining polymer-tissue interactions.

Conclusion

This review has highlighted the versatility and utility of gelatin-based controlled release
systems in various biomedical applications. Different bioactive molecules can be loaded into
gelatin carriers while retaining their inherent biological activity. Additionally, gelatin-based
matrices are able to protect loaded biomolecules from degradation and release them over
extended time periods. By altering the crosslinking density of the gelatin hydrogel carrier, the
period of biomolecule release can be regulated, enabling investigators to tailor this
biomaterial with the optimal release characteristics required for each individual application.
However, it still requires modification (e.g., crosslinking, grafting) to be used as a carrier for
a broader range of bioactive molecules in novel various scaffolds and carriers.