Microencapsulation for Therapeutic Applications

Ph.D.Thesis Defense by Nikhil Dhoot Department of Chemical Engineering Drexel University, Philadelphia PA 19104 Advisor: Prof. Margaret Wheatley March 19, 2002

Outline

Microencapsulation Alginate Liposomes Microencapsulated liposomes for protein delivery Spinal cord injury Encapsulation of genetically engineered fibroblasts for treatment of spinal cord injuries Modification of microcapsule surface Conclusions Recommendations

Microencapsulation

First studied in the food industry and in the development of carbonless paper Use in drug delivery pioneered almost 40 years ago Several systems developed for the encapsulation of different therapeutic agents as well as for encapsulation of cells Variety of polymers used: Alginate, agarose, gellan gum, gelatin, chitosan, PLGA, polyvinyl alcohol, collagen, polyacrylamide Various methods used: Emulsification, ionotropic gelation, thermal gelation, in situ polymerization, interfacial polymerization

Microcapsules for Drug Delivery

Kinetics of drug release can be altered Duration of release can varied over great periods of time Surface modification can enable targeting to specific sites Most methods of preparation utilize organic solvents Aggregation of proteins and loss of protein activity has been reported Sterilization of microcapsules also poses a problem Water soluble polymers such as alginate present an attractive choice for protein delivery

Choice of Polymer and Method of Microencapsulation

The polymer should be

Non-toxic Biocompatible Not interact with the drug or protein

The microencapsulation method should be mild and should not involve the use of

High temperatures Harsh solvents

Alginate

Naturally occurring linear polysaccharide 1,4 linked -L-guluronic acid and -D-mannuronic acid Non-toxic reactants and mild conditions for encapsulation Crosslinks in the presence of multivalent cations Easy control of physico-chemical properties of capsules Easy surface modification with proteins Established use for encapsulation of flavors, enzymes and cells

Mannuronic Acid

Guluronic Acid

Eggbox model for crosslinking of alginate with calcium ions

Set-up for Microcapsule Preparation by Ionotropic Gelation

Size of microcapsules prepared is about 300-400 m

Coating of Alginate Gels

Carboxylate groups on the surface are available to react with other cations Coating of the gel with a polycation such as poly-L-lysine and poly-L-ornithine can act as a size exclusion membrane The coating can serve as a barrier to exclude immunogenic substances Coating can also provide strength to the capsule Characteristics of the coating can be altered by varying the conditions of the coating process

Liposomes
Artificial vesicles made from phospholipids; bilayered in structure Used as a model for biological membranes Widely studied for drug delivery Taken up by the RES

Cross sectional view

Source: Biochemistry by Lehninger

Classification

SUVs (10-100 nm) LUVs (100-1000 nm)

MLVs (100 nm-20 m) MVVs (100 nm-20 m)

Liposomes Encapsulated in Alginate

Immune Cells and Antibodies

Liposomes Alginate Matrix

Semipermeable membrane

Previous Studies

Encapsulation of liposomes in collagen, nylon, acaciagelatin (Weiner et al, 1985; Nixon et al, 1989; Dong et al, 1993) Encapsulation of liposomes in alginate for protein delivery (Wheatley et al, 1987; Kibat et al, 1990; Cohen et al, 1991) Liposomes encapsulated in alginate have been shown to elicit higher immune responses (Cohen et al, 1991) Encapsulation of liposomes in alginate results in an initial burst of protein release Also, the possibility of obtaining pulsatile release has been demonstrated

Objectives

To encapsulate liposomes in alginate microcapsules and to optimize the encapsulation procedure To determine the effect of different crosslinking ions on protein release To determine the effect of different types and compositions of liposomes on the release profile To investigate the possibility of interaction between the liposomes and alginate and to determine the mechanism of any such interaction

Methodology

Liposomes of different types (LUVs and MLVs) and compositions were prepared Liposomes were encapsulated in 1.5% (w/v) alginate crosslinked with different cations (Ca2+, Al3+, Ba2+) Microcapsules were coated with a 0.5 mg/ml poly-Lornithine solution followed by coating with 0.12% (w/v) alginate Release of protein (FITC-BSA) from microencapsulated liposomes and from non-encapsulated liposomes was monitored

Effect of Crosslinking Ions on Protein Release (PC/Chol Liposomes)

100 90
Cumulative Release (%)

80 70 60 50 40 30 20 10 0 0 5 10 15 Time (days) 20 25 30
encapsulated in Ca-alginate encapsulated in Al-alginate encapsulated in Ba-alginate non-encapsulated

Effect of Crosslinking Cations

Ca2+ ion 0.97

Ba2+ ion 1.35

Al3+ expected to give three dimensional binding structure Small size of Al3+ ion 0.5 Less compact structure compared to Ba-Alginate

PC/Chol (7:3) liposomes encapsulated in Ca2+ crosslinked alginate; a. Freshly prepared b. After 2 days c. After 10 days

Scale Bar = 500 m

PC/Chol (7:3) liposomes encapsulated in alginate after 10 days; a. Crosslinked with Al3+ b. Crosslinked with Ba2+ Similar behavior was observed in the case of PC/PG/Chol liposomes
Scale Bar = 500 m

Effect of Liposome Composition (PC Liposomes)

100 90
Cumulative Release (%)

80 70 60 50 40 30 20 10 0 0 5 10 15 Time (days) 20 25 30
encapsulated in Ca-alginate non-encapsulated liposomes

Effect of Liposome Composition (DOPC Liposomes)

100 90
Cumulative Release (%)

80 70 60 50 40 30 20 10 0 0 5 10 15 Time (days) 20 25 30
DOPC encapsulated in Ca-alginate DOPC/Chol encapsulated in Ca-alginate non-encapsulated DOPC non-encapsulated DOPC/Chol

Effect of Liposome Type (MLVs)

100 90
Cumulative Release (%)

80 70 60 50 40 30 20 10 0 0 5 10 15 Time (days) 20 25 30
Encapsulated PC/Chol MLV's Encapsulated PC/PG/Chol MLV's non-encapsulated PC/Chol MLV's non-encapsulated PC/PG/Chol MLV's

Interaction Between Liposomes and Alginate

Differential Scanning Calorimetry performed on liposomes showed a decrease in transition enthalpy indicating insertion of alginate into the lipid bilayer
0.2

DOPC DOPC in 1.5% alginate

Heat Flow (mW)

0 -0.2 -0.4 -0.6 -0.8 -30 -25 -20 -15 Temp (C)

-10

-5

Interaction Between Liposomes and Alginate

Liposome Composition
DOPC DOPC/Chol

Without alginate
Tm= -16.73C, H= 420.7 J/g Tm= -20.5C, H= 14.50.5 J/g

In presence of 1.5% alginate

Tm= -16.93C, H= 34.50.4 J/g Tm= -20.62C, H= 9.750.3 J/g Tm= 44.5C, H= 6.51 J/g

DPPC (from Cohen Tm= 44.58C, et al, 1991) H= 22.93 J/g

Conclusions

Release of protein is faster from liposomes encapsulated in alginate than from non-encapsulated liposomes Crosslinking ions have a significant effect on the release of protein from microencapsulated liposomes The initial burst of protein release can be prevented by

Crosslinking alginate with Ba2+ ions Encapsulation of LUVs without cholesterol Encapsulation of MLVs

DOPC and DOPC/Chol liposomes exhibit a delayed pulse of protein release after encapsulation in alginate Alginate is inserted in to the lipid bilayer leading to an increase in permeability

Spinal Cord Injuries

Approximately 10,000 people sustain new spinal cord injuries every year in the United States alone Majority of injuries occur in young people, less than 30 years of age Lack of effective treatment leads to a need for life long care and rehabilitation The devastating effects of spinal cord injury are due to

Death of neurons that cannot be replaced Failure of surviving neurons to regenerate their axons Inhospitable environment produced by the injury

Approaches to Treatment

Transplants of peripheral nerves (Richardson et al, 1980) Transplants of Schwann cells (Li et al. 1994) Transplants of fetal cells (Jakeman et al, 1991) Application of neurotrophic factors (Tezlaff et al, 1994; Diener et al, 1994) Transplants of genetically engineered cells

Neuronal cells (Snyder et al, 1997) Stem cells (Liu et al, 1999) Fibroblasts (Liu et al, 1999) Collagen (Grill et al, 1997) Matrigel (Xu et al, 1997) Polyethersulfone hollow fibers (Hoffmann et al, 1993)

Transplants of encapsulated cells

Neurotrophic Factors

Neurotrophic factors like NT3 and BDNF can promote regeneration and recovery of function These factors should be delivered effectively to the site of the injury Transplantation of genetically engineered cells that produce the neurotrophic factors is one of the most promising approaches These approaches are expected to lead to development of protocols for clinical trials in the near future

Genetically Engineered Fibroblasts

Retrovirus constructs of BDNF have been prepared and used to genetically engineer primary skin fibroblasts When grafted into the injured spinal cord of rats, these cells

survive in vivo rescue axotomized neurons and promote regeneration contribute to recovery of locomotor function

Grafting of these allografts in animal models requires strict immunosuppression protocols to prevent rejection These problems can be overcome by encapsulating the cells in a semipermeable polymer matrix

Cells Encapsulated in Alginate

Immune Cells and Antibodies Nutrients

Protein

Waste

Cells

Alginate Matrix

Semipermeable membrane

Objectives

To encapsulate BDNF-producing fibroblasts (Fb/BDNF) in alginate and examine survival of and transgene expression in the encapsulated cells in vitro. To test the in vivo efficacy of the encapsulated cells when transplanted into an injured spinal cord without immunesuppression To evaluate functional recovery in animals receiving transplants of encapsulated cells To develop a microcapsule surface that is permissive for the for the growth of neurons

In vitro Growth of Encapsulated Cells

Fb/BDNF fibroblasts were encapsulated in 1.5% (w/v) alginate at a concentration of 3106 cells/ml and the microcapsules were coated with poly-L-ornithine (Scale bar = 100 m)

a
Immediately after encapsulation

b
3 days

7 days

12 days

Reporter Gene Expression in Encapsulated Cells

After 2 days

After 7 days

Scale bar = 250 m

After 4 weeks

Reporter Gene Expression in Encapsulated Cells after Storage

Encapsulated Fb/BDNFwere frozen in freezing medium used for non-encapsulated cells and stored in liquid nitrogen. The frozen capsules were then thawed and transferred to culture. Scale bar = 250 m

A
2 days after thawing

B
3 weeks after thawing

Bioactivity of Secreted BDNF

Embryonic chick DRG (dorsal root ganglion) explant assay

A. Unconditioned media B. Media containing human recombinant BDNF Conditioned media from cultures of C. Empty capsules D. Encapsulated Fb/BDNF E. Unencapsulated Fb/BDNF

Scale bar = 100 m

Spinal Cord Injury Model

White matter

Lesion site

Grey matter
Encapsulated Fb/BDNF were transplanted into the lesion area in the absence of immunesuppression

Grafting of Encapsulated Cells into the Injured Spinal Cord

Reporter Gene Expression in Explanted Capsules

Scale bar = 100 m

A
Scale bar = 500 m

A
Capsule harvested at 4 weeks

B
Scale bar = 100 m

B
After 1 week in culture

Host Immune Response

GFAP staining for presence of astrocytes at the injury site 4 weeks after transplantation of encapsulated Fb/BDNF (b) or empty capsules (a) Scale bar = 200 m

Host Immune Response (Contd.)

ED-1 staining for presence of glial cells and macrophages at the injury site 4 weeks after transplantation of encapsulated Fb/BDNF (b) or empty capsules (a) Scale bar = 200 m

Stains for Axonal Growth

RT-97 staining for axonal growth in the lesion area after transplantation of encapsulated Fb/BDNF at 2 weeks (a) and 4 weeks (b). The total amount of staining increased from 244 m at 2 weeks to 2000 m at 4 weeks. Scale bar = 200 m.

Stains for Axonal Growth (contd.)

At 4 weeks post-transplantation, axons immunoreactive for MAP-1B (a) and dendrites immunoreactive for MAP-2 were observed closely associated with the outer wall of the capsules. Staining for these markers was not observed at 2 weeks after transplantation. Scale
bar = 100 m.

Behavioral Analysis

a
Healthy Rat

Functional recovery was evaluated using the cylinder test Surgery caused the right forelimb to be impaired Total movements of the right forelimb were recorded at different times after surgery Four groups of animals were studied:

b
Injured Rat

Encapsulated Fb/BDNF Empty capsules Fb/BDNF in gelfoam with CSA Fb/BDNF in gelfoam without CSA

90 80 70
Total use of right forelimb (%)

60 50 40 30 20
Fb/BDNF in gelfoam + CSA

10 0 Baseline Week 1 Week 2 Week 3

Empty Capsules Fb/BDNF + Capsules Fb/BDNF in gelfoam no CSA

Week 4

Week 5

Functional recovery of right (impaired) forelimb use after transplantation of encapsulated Fb/BDNF. The difference in recovery between groups receiving Fb/BDNF capsules and empty capsules is statistically significant (p<0.05) at weeks 3 and 5 and that between Fb/BDNF capsules and Fb/BDNF without immunesuppression is statistically significant (p<0.05) week 3 onwards.

Modification of Microcapsule Surface

Laminin is an extracellular matrix glycoprotein Laminin is known to enhance neurite outgrowth in vitro Several domains believed to promote neurite extension have been isolated Carboxylate groups on the alginate provide the possibility of creating a growth permissive surface YIGSR peptide was covalently conjugated on to the alginate via a water soluble carbodiimide Gels prepared from alginate-YIGSR conjugate were studied for their ability to support neurite outgrowth from NB2a neuroblastoma cells in vitro

Schematic of YIGSR-Alginate Gels

Cell adhesion through a 68kD non-integrin protein

No cellular adhesion YIGSR peptide Unmodified alginate gel YIGSR-alginate gel

Neurite Outgrowth on YIGSR-Alginate

Neurite outgrowth of NB2a neuroblastoma cells on YIGSR-alginate gels with 2 mg peptide/g alginate. Cells were allowed to attach on the modified alginate surface for 24 hours (A) and then were treated with 10 M dibutyryl cyclic AMP to induce differentiation. After 7 days, a large number of cells demonstrated extensive neurite outgrowth (B-D). Scale Bar = 100 m

Neurite Outgrowth on YIGSR-Alginate (Contd.)

Conclusions

Genetically engineered fibroblasts grow and secrete BDNF in an active form in vitro after encapsulation in alginate Encapsulated cells survived and continued to express the transgene in vivo without immune suppression Transplants of encapsulated cells induced axonal regeneration at the injury site which appeared to increase with time while eliciting a non-specific immune response Significant functional recovery was observed in animals receiving transplants of encapsulated cells YIGSR-alginate conjugate can be used to provide a surface that is permissive for axonal growth.

Acknowledgements

Dr. Wheatley Dr. Fischer, Dr. Murray and Chris Tobias at MCP Chemical Engineering Faculty and Committee Members Graduate students in my lab and in Chemical Engineering Dan Luu and George Carenzo My wife, Arati

Advantages

Disadvantages

Both hydrophobic and hydrophilic drugs can be entrapped Reduction in toxic effects Prolonged drug retention Versatility due to the wide spectrum of available phospholipids and the different types of liposomes Generally, do not interact with the drug or alter it in any form

Tend to aggregate or lose entrapped drug during storage Cannot be sterilized by heat or radiation Lack of consistency between preparations makes clinical use difficult Small lab scale preparations are very difficult to scale-up

They are taken up by the RES

Effect of Crosslinking Ions on Protein Release (PC/PG/Chol Liposomes)

100 90
Cumulative Release (%)

80 70 60 50 40 30 20 10 0 0 5 10 15 Time (days) 20 25 30
encapsulated in Ca-alginate encapsulated in Al-alginate encapsulated in Ba-alginate non-encapsulated liposomes

Recommendations

Co-encapsulation of liposomes of different compositions Determine activity of protein released Encapsulation of liposomes in hydrogels that do not require cations for crosslinking In vivo evaluation of the microencapsulated liposome system Determine the source of axons in the transplant area Investigate the in vivo downregulation of the transgene Examine long term effects of the encapsulated cells in the spinal cord Further studies on creating a growth permissive surface Combination delivery of different neurotrophic factors or antibodies to inhibitors of neuronal growth