Engineering of Gelatin for Drug Delivery Applications...Engineering of Gelatin for Drug Delivery Applications Dr. K. Subramanian, Professor Department of Biotechnology Bannari Amman

Engineering of Gelatin for

Drug Delivery Applications

Dr. K. Subramanian, Professor

Department of Biotechnology

Bannari Amman Institute of Technology

Sathyamangalam-638401, Erode-Dt

Tamilnadu, INDIA

Email: [email protected]

Gelatin (A/ B)-a translucent, colorless, brittle, flavorless denatured protein-obtained by acid (A) or base (B)hydrolysis of collagens from animal skin & bone .

Gelatin A (pI=7–9) and B(pI=4-5) – have different isoelectric point , mol.wt, amino acid composition, and viscosities.

It is a heterogeneous mixture of single or multi-stranded random coiled poly- peptides, each with extended left-handed proline helix having 300 to 4000 amino acids. versatile biomaterial commonly used as a gelling agent in food and pharmaceuticals

Gelatin-important features

8 July 2016 Asiapharma 2016, 11-13 July 2016, Kaulalumpur 2

Intrinsic features- great potential to modify at the level of amino acids, low level of immunogenicity and cytotoxicity, FDA approval as a clotting agent and exudate absorbing construct, hydrogel formation and biodegradability.

-Asp-Lys -Ala-Gly-Pro-Arg-Gly-Glu-4Hyp-Gly-Pro-Tyr-

Gelatin-features

NH CH C

CH3

NH

O

C

O

N

C NH

O

CH

CH2

CH2

CH2

NH

C

C

NH2

NH2

+

NH

O

CH2 C NH

O

CH C

CH2

CH2

C

O-

O

O

N

OH

CH

OH

NH CH C

OH

N

C

O

NH CH CH

CH3

C O

NH

OH

CH

C

OH

O

C

O

CHNH

CH2

CH2

CH2

CH2

NH2

CCH

O

NH

CH2

C

O-

O


Gly 21%, Pro 12%, HyP 12%, Glu 10%, Ala 9%, Arg 8%,

Asp 6%, Lys 4%, Ser 4%, Leu 3%, Val 2%, Phe 2%, Thr 2%,

Ile 1%, HLys 1%, Met and His <1% and Tyr <0.5%

Gelatin-a polyampholyte , ~12% -vely charged due to Glu &

Asp, ~13% +vely charged due to Arg and Lys residues , and

~11% of its chain hydrophobic due to Ile, Met, Leu and Val

residues, in an approximate ratio of 1:1:1 respectively. Pro,

Gly & HPro occurring as a sequence of repeating triplet

constitute the rest of gelatin

Gelatin-composition of amino acids


Use of gelatin,a safe excipient approved by FDA of US,

in various pharmaceutical dosage forms dates back to

the early nineteenth century or before.

Today, million pounds of gelatin is used annually as

hard and soft elastic capsules, tableting, tablet coating,

granulation, encapsulation, and micro-encapsulation.

The good adhesive properties and easy shattering of its

agglomerates are its additional advantages.

Features of gelatin


pI values of gelatin can be modified to get either a -vely

charged acidic gelatin, or a +vely charged basic gelatin

at physiological pH to facilitate electrostatic

interactions with charged biomolecule ( protein,

plasmid DNA etc)

Its heterogeneity in mol.wt, high water solubility, poor

structural & thermal stability- make it a poor carrier to

achieve controlled and prolonged drug release

Features of gelatin


But its primary structural features offers many chemical modification that enable the design of different drug carrier systems, such as microparticles and nanoparticles, fibers and hydrogels for controlled release.

Gelatin microparticles can serve as vehicles for cell amplification and for delivery of large bioactive molecules & its nanoparticles are better suited for intravenous delivery or for drug delivery to the brain.

Gelatin fibers contain a high surface area-to-volume ratio

Gelatin hydrogels can trap drugs between the polymer's crosslink gaps, allowing them to diffuse into the blood stream. Gelatin forms complexes with different drugs

Features of Gelatin


Nanoparticle and microparticle formation

Chemical Crosslinking –PEG-dialdehyde, diisocyanate, genipin, glutaraldehyde etc

Enzymatic crosslinking- using transglutaminase

PEGylation

Thiolation via ε-amino groups

Blending with other polymers

IPN formation with appropriate polymer

Grafting

Conjugation with synthetic polymers and antibodies

Anionization/cationization

Entrapping green synthesized Ag nanoparticles

Converting carboxylic group to amido group

Introducing hydrophobic alkyl group via pendant amino group

Gelatin modifications


Alters biofunctional properties such as solubility, swellability, biodistribution, biocompatibility, targetability, biodegradability, hydrophilicity/ hydrophobicity, morphology, and stability of the formulations

Improve its flexibility to overcome challenges in finding ideal carrier systems that enable specific, targeted and controlled release in response to body demands.

Effect of gelatin modification


(i) isophorone diisocyanate (IPDI) &

(ii) isocyanate terminated oligomeric poly(ethylene

glycol) (PEG-600) ( ICTPE)-prepared by reacting IPDI

and PEG-600 in DMSO

These were used as crosslinkers to modify gelatin

properties because of the less toxicity of IPDI compared to

other diisocyanate crosslinkers and biocompatibility of

PEG moiety in ICTPE

Diisocyanate crosslinker


ICTPE Crosslinked gelatin Feed Composition for prepolymer (ICTPE) (mole)

Gelatin (g)

PEG-600 ( 103) IPDI ( 103)

GEPI-5 0.4483 0.8966 2

GEPI-10 0.896 1.793 2

GEPI-20 1.799 3.599 2

GEPI-30 5.398 5.398 2

Diisocyanate crosslinked gelatins

IPDI crosslinked gelatins using 1, 2, 5, 10, 20, and 30 wt% of IPDI with reference to

the weight of gelatin taken were designated as GEIP-1, GEIP-2, GEIP-5, GEIP-10,

GEIP-20, and GEIP-30,


Free pendant hydroxyl, amino, carboxyl, etc. of gelatin react with isocyanate in the rate order: –NH2 > –OH > –COOH to form urea (–NH–C(O)–NH–), urethane (–NH–C(O)–O–), and carbamate (–NH–C(O)–O–C(O)) links, respectively.

-NH2 + -NCO ---- -NH-C(O)-NH- (Urea)

-OH + -NCO ---- -O-C(O)-NH- (Urethane)

-COOH + -NCO --- -NH-C(O)-O-C(O)--- (Carbamate, Unstable)

Since the carbamate is unstable, it is converted into amide bond with release of carbon dioxide

(–NH–C(O)–O–C(O)-- - –NH–C(O)–NH– + CO2

Isocyanate reactions with gelatin

8 July 2016 Asiapharma 2016, 11-13 July 2016, Kaulalumpur

12

IPDI crosslinked gelatin


CH3

CH3

CH3NCO

N C O

IPDI

CO2+3

OH Gelatin COOH

NH2

NH2

CH2

CH3

CH3

CH3CH2NHC

O

NH

NH

CH3

CH3

CH3CH2 NH C

O

Gelatin

NH

O

C O

NH

NH

CH3

CH3

NH C

O

NH Gelatin

NH

O

C O

C O C O

C O

DMSO/ 35 °C / N2 / 24h

Cross linked gelatin

2

ICTPE crosslinked gelatin


OH Gelatin COOH

NH2

NH2

DMSO/ 35 °C / N2 / 24h

ICTPE Cross linked gelatin

2

CH3

CH3

CH3NCO

N C O

IPDI

2 + OH CH2 CH2 O CH2 CH2 OH

n

O CH2 CH2 O CH2 CH2 OCH3

CH3

CH3NCO

NH C

O

C

CH3

CH3

CH3NH

O

N C O

n

O CH2 CH2 O CH2 CH2 OCH3

CH3CH3

NH C

O

C

CH3

CH3

CH3NH

O

NH

NH

C

C

O

NH Gelatin

NH

O

C O

NHC

O

NHCH3CH2 NH C

O

Gelatin

CH3

O

NH

C

NH

OO CH2 CH2 O CH2 CH2 O

CH3

CH3

CH3

NH C

O

C

CH3

CH3

CH3NH

O

NH

C

NH

O

CH3

NHO CH2 CH2 O CH2 CH2 ONH C

O

C

O

CH3

CH3

CH3

NH

C

NH

O

O

n

nn

DMSO/ 35 °C / N2 / 24h

CO2+

PEG-600

ICTPE

Reaction of IPDI and ICTPE generates urethane, amide, and urea linkages- it is anticipated that they will display different mucoadhesive and swelling characteristics, which may influence the drug release characteristics.

Moreover, the PEG spacer may minimize the loss in the flexibility and swellability of the gelatin due to crosslinking.

The crosslinked gelatins carriers were evaluated by in vitro release studies THP and 5-FU tablets in SIF & SGF.

Structural effect of drugs on their release features were also tested for a typical crosslinked gelatin carrier taking 5-FU, AMP Na & THPas specific examples.

DIISOCYANATE CROSSLINKED

GELATIN


Subramanian K, Vijayakumar V. Polymer Bulletin,

2013, 70(3): 733–753

V.Vijayakumar, K. Subramanian, Saudi Pharmaceutical

Journal,2014, 22: 43-51.

IPDI and ICTPE Crosslinked gelatin Synthesis

and characterization - NMR, FT-IR, XRD etc


(A) TG and (B) DTG traces of gelatin (a) and

crosslinked gelatins [GEIP-5 (b), GEIP-10 (c)]

200 400 600 800

0

20

40

60

80

100

Re

sid

ua

l W

eig

ht

(%)

Temperature( C)

GE(a)

GEIP-5(b)

GEIP-10(c)

a

b

c

(A)

100 200 300 400 500 600 700 800 900

-16

-14

-12

-10

-8

-6

-4

-2

0

De

riv

ati

ve

We

igh

t %

(%

/min

)

Temperature (°C)

GE(a)

GEIP-5(b)

GEIP-10(c)

c

b

a

(B)


TGA (A) and DTG (B) traces of, (a) GE, (b)

GEPI-5, (c) GEPI-10 and (d) GEPI-20


100 200 300 400 500 600 700 800 900

0

10

20

30

40

50

60

70

80

90

100

We

igh

t (%

)

Temperature (°C)

a) GE

b) GEPI-5

c) GEPI-10

d) GEPI-20

a

bcd

(A)

100 200 300 400 500 600 700 800

Temperature(oC)

(a) GE

(b) GEPI-5

De

riv

ati

ve

We

igh

t (%

/C

)

(c)GEPI-10

(d)GEPI-20 (B)

DSC thermograms of gelatin (a), regenerated gelatin (b)

and crosslinked gelatins GEIP-1(c); GEIP-5(d); & GEIP-10

(e)


50 100 150 200 250 300 350 400

15

16

17

18

19

20

21H

ea

t fl

ow

en

do

up

(m

W/g

)

Temperature(oC)

a

b

c

d

e

DSC traces of , (A) gelatin and crosslinked

gelatins and (B) drugs and tablets


60 80 100 120 140 160 180 200 220 240 260 280 300 320

19.5

20.0

20.5

21.0

21.5

22.0

22.5

23.0

He

at

flo

w e

nd

o u

p(m

W)

Temperature(oC)

GE(a)

GEPI-5(b)

GEPI-10(c)

GEPI-30(d)

(a)

(b)(c)

(d)

(A)

60 80 100 120 140 160 180 200 220 240 260 280 300 320

20

30

40

50

60

70

He

at

flo

w e

nd

o u

p(m

W)

Temperature( C)

5FU(a)

GEPI-10F(b)

THP(c)

GEPI-10T(d)

AMP(e)

GEPI-10A(f)

a

b

c

de

f

(B)

Effect of wt % of IPDI on B) % acetone insoluble fraction

(A) and residual amino group (b) in aged IPDI cross linked

gelatin


0 5 10 15 20 25 30

0.0001

0.0002

0.0003

0.0004

0.0005

0.0006

Re

sid

ua

l a

min

o g

rou

p(e

qu

/g)

Wt % of IPDI

(A)

0 5 10 15 20 25 30

30

35

40

45

50

55

60

65

a) SGF

b) SIF% R

es

idu

al W

eig

ht

aft

er

1 w

ee

k

% weight of IPDI

a

b

(B)


Effect of ICTPE wt% on residual amino group (A) and% acetone insoluble fraction(B) of

aged cross linked gelatin in SGF (a) and SIF (b), and biodegradation of GEPI-5 and

GEPI-20 with time (C) in SGF and SIF.

5 10 15 20 25 30

4.0

4.5

5.0

5.5

6.0

6.5

7.0

Re

sid

ua

l a

min

o g

rou

p(e

qu

/g)

(x

10

7)

Weight % of ICTPE

(A)

0 2 4 6 8 10 12

50

60

70

80

90

100

SGF

GEPI-5

GEPI-20

We

igh

t(%

)

Time(days)

SIF

GEPI-20

GEPI-5

(C)

A)Optical microscopic photographs and B)Histogram - % cell

viability of NIH 3T3 cell growth on GEIP-5


0

20

40

60

80

100

0.025 0.05 0.1 1 10%

Cell

Via

bilit

y

Concentration (mg/mL)

Degree of swelling of IPDI cross-linked gelatins (GEIP-

2(a), GEIP-5(b),GEIP-10(c), GEIP-20 (d) and GEIP-

30(e)) in SGF (A) and SIF (B) at 37 C


0 15 30 45 60 75 90 105 120 135 150 165 180 195 210

0

2

4

6

8

10

12

14

16

De

gre

e o

f s

we

llin

g

Time(min)

a

b

c

d

e

(A)

0 15 30 45 60 75 90 105 120 135 150 165 180 195 210

0

2

4

6

8

10

12

14

16

De

gre

e o

f s

we

llin

g

Time(min)

a

b

c

d

e

(B)

C

Release profiles -tablets , 5-FU with IPDI cross linked

gelatin in SGF (A) and SIF (B) & THP, FU & AMP with

GEIP-30 (C) at 37 C

0 15 30 45 60 75 90 105 120 135 150 165 180 195 210

0

10

20

30

40

50

60

70

80

90

100

% D

rug

re

lea

se

d

Time(min)

SGF

GE

GEIP-1

GEIP-2

GEIP-5

GEIP-10

GEIP-20

GEIP-30

(A)

0 15 30 45 60 75 90 105 120 135 150 165 180 195 210

0

10

20

30

40

50

60

70

80

90

100

SIF

GE

GEIP-1

GEIP-2

GEIP-5

GEIP-10

GEIP-20

GEIP-30

% D

rug

re

lea

se

d

Time(min)

(B)

0 50 100 150 200

0

20

40

60

80

100

% D

rug

re

lea

se

d

Time(min)

SGF SIF

THP THP

AMP AMP

FU FU


Swelling profiles of ICTPE crosslinked gelatins GEPI-5(a),

GEPI-10(b), GEPI-20 (c) and GEPI-30(d) in SGF (A) and

SIF (B) at 37 C

0 30 60 90 120 150 180 210

0

2

4

6

8

10

12

14

16

De

gre

e o

f s

we

llin

g

Time(min)

(a)

(b)

(c)

(d)

SGF(A)

0 15 30 45 60 75 90 105 120 135 150 165 180 195 210

0

2

4

6

8

10

12

14

16

De

gre

e o

f s

we

llin

g

Time(min)

(a)

(b)

(c)

(d)

SIF(B)


c

Release profiles of, THP with (a) GEPI-5, (b) GEPI-10, (c) GEPI-20

and (d) GEPI-30 in SGF (A) and SIF (B) & THP, AMP and 5-FU in SGF

and SIF with GEPI-30 (C) at 37 °C

0 50 100 150 200 250 300 350

0

10

20

30

40

50

60

70

80

90

100

% D

rug

re

lea

se

d

Time(min)

SGF

GEPI-5

GEPI-10

GEPI-20

GEPI-30

(A)

0 50 100 150 200 250 300 350

0

10

20

30

40

50

60

70

80

90

100

SIF

GEPI

GEPI-5

GEPI-10

GEPI-20

GEPI-30

Time(min)

% D

rug

re

lea

se

d

(B)

0 50 100 150 200 250 300

0

20

40

60

80

100

% D

rug

re

lea

se

d

Time(min)

SGF SIF

THP THP

AMP AMP

5FU 5FU


Drug release was modeled using the Korsemeyer-

Pappas empirical equation

Mt/M∞ = ktn

where Mt/M∞ - fractional release at time t, k- rate

constant & n - diffusional exponent.

For IPDI and ICTPE crosslinked gelatin the n values were

in the range 0.6–0.9-non-Fickian

Drug release mechanism


Crosslinking of gelatin with IPDI & ICTPE-decreased its water solubility, biodegradability, and swellability and enhanced amorphous nature

In vitro release profiles of 5-FU -decreased drug release rate with increased crosslinking. The 5-FU release rate is more in SGF than in SIF

Comparison of typical release profiles for AMP sodium, THP, and 5-FU for a typical crosslinked carrier indicated only marginal differences in release rates due to variation in hydrophobicity / hydrophilicity of drugs

Non-Fickian (anamalous transport,n=0.6 to 0.9)release mech.

Conclusions


The cross linked gelatin - biodegradable in SIF and SGF & biodegradability decreased with increased degree of cross linking. Drug release by mainly by swelling-diffusion. Minor gelatin degradation in SGF and SIF may also contribute for the drug release rate.

The results suggested that the cross linked gelatin might be a promising vehicle for sustained release preparations in oral therapy.

This material can also be used in tissue engineering as scaffold in medical field because of its biocompatibility, structural integrity and biodegradability.

Conclusions



Acknowledgement

The financial support by Department of Biotechnology(DBT), Government of

India to execute this research work is thankfully acknowledged.

The author is very thankful to Dr. Vijayakumar V, Assistant Professor, Bannari

Amman Institute of Technology, India for carrying out this research work

with the DBT fellowship.

The permission and financial support extended by the Management of Bannari

Amman Institute of Technology, India to participate in Asiapharma 2016 are

also acknowledged with thanks

END

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Engineering of Gelatin for Drug Delivery Applications...Engineering of Gelatin for Drug Delivery Applications Dr. K. Subramanian, Professor Department of Biotechnology Bannari Amman