SILK-BASED DELIVERY SYSTEMS OF BIOACTIVE MOLECULES - PowerPoint Presentation

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SILK-BASED DELIVERY SYSTEMS OF BIOACTIVE MOLECULES

Presented by:

Sk.Asma

ShahedaSlide2

? ?? ? What is a silk based delivery

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CONTENTSIntroduction Sources of silk proteins 1.Natural -Silk worm fibroin

-Spider silk fibroin

2. Recombinant -Silk worm variants

-Spider silk variants

Advantages of silk proteins

Preparation of

Sericin

free silk solution

Biosynthesis of recombinant spider silk proteins

- Design, construction, and cloning of the genes, and

- Expression and purification of the protein polymers.Slide4

Applications of Silk worm silk protein for drug delivery with various examples a. Scaffolds b. Silk films c.

Nanofibres

d. Microspheres

e.

Nanoparticles

f.

Microneedles

g.

Hydrogels

h. Coatings

Applications of recombinant Spider silk for drug and gene delivery with various examples

a. Reconstituted spider silk as microspheres

b. Spider silk-

polycation

block copolymers

c. spider silk-

polycation

functional peptide

multiblock

copolymers

Release of drug from silk matrix and fate of silk fibroin

Conclusion

References

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INTRODUCTION:Silk based delivery systems deals with the use of silk protein as a polymer for various drug delivery systems.

Silks are biodegradable, biocompatible, self-assembling proteins that can be tailored via genetic engineering to contain specific chemical features, offering its utility for drug and gene delivery.

This topic focuses on the biosynthesis of silk-based polymer

systems and related silk protein drug delivery.

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SOURCES OF SILK PROTEINS: 1.Natural silk proteins:

Produced by a variety of insects and spiders.

Biodegradable and possess high mechanical properties.

Functions include development(cocoons), prey capture(spider webs), to safety lines(Spider dragline).

Have their applications in biomedical suture, biomaterial culture and tissue engineering.

Silk worm fibroin:

It has its own applications in biomedical suturing for decades and in textile production for clothing for centuries

.

Structure:

Silk is a

continuos

strand of two filaments cemented together forming the cocoon of silk worm.

Silk filament –double strand of fibroin-held by Silk

sericin

.

Silk fibroin -glycoprotein and composed of 2

equimolar

protein subunits,

-light and heavy chain fibroins linked by disulphide bonds.

Best example of

β

-pleated structure

.

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Beta pleated structure of the silk

Amino acid composition of the fibroinSlide8

b. Spider silk fibroin:Primary structure is its amino acid sequence with highly repetitive glycine and alanine

blocks, which are referred to

as block copolymers.

Protein composition in the primary structure

Large Hydrophobic blocks with Small hydrophilic blocks with more highly conserved sequences consisting complex sequences that consist

Of short side-chain amino acids such as

aminoacids

with bulkier side-chains

Glycine

and

alanine

. and charged aminoacids.Show α-helices in the solutionAnd β-sheet structures in the assembled formThus a primary structure possess a amphiphilic composition as that of surfactants and biological membranes. During secretion from the spinning duct, repetitive sequences undergo intra and inter-molecular interactions resulting in the formation of secondary, tertiary, quaternary structure.Slide9

a. Secretion of silk thread from the spinning duct b. Structure of spider silk

fibroin Slide10

Non-protein composition of spider silk: Compound(s)

Uses

Mechanism

Sugars, lipids, ions, and pigments

Act as protection layer

in the final

fibre

-----------------

Pyrollidine

Keeps thread moist

Due to its hygroscopic properties

Potassium hydrogen phosphate

Make the silk acidic and protect from fungi and bacteria that digest the proteinReleases protons in aqueous solution, resulting in pH-4Potassium nitratePrevent from denaturing in the acidic medium-------------Slide11

DIFFERENCES BETWEEN SILKWORM AND SPIDER SILK :-

SILK

WORM SILK

SPIDER SILK

Molecular level

Large amount of

sericin

is present

Sericin

is absent

Proteins responsible for

fibrillar

structures Called as fibroins and contains light and heavy fibroinsCalled as fibroins or spidroins specifically and contains light and heavy counter-partsMechanical propertiesWeaker and less extensible Stronger with high extensible propertiesSpinning conditionsEither strong or elasticBoth strong and elasticSlide12

2.Recombinant silk proteins:a. Silk worm variants: Silk like repeats of (GAGAGS), elastic block copolymers, Silk-elastin

-like proteins (SELP)

Results:

Enhanced gene expression was reported in target cells up to 10 fold, when compared to viral injection without the SELP.

With insertion of partial collagen and

fibronectin

sequences, cell-adhesive ability was increased.

Films made from recombinant silk proteins had six-fold higher activity than original silk fibroin.

b. Spider variants:

Spider silk sequence was modified to contain

methionines

adjacent to

polyalanine sequence, controlled self assembly of beta-sheet structures in silk.Modified spider silk, which was 15mer of [SGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGT] derived from the spidroin was bioengineered to include arginyl-glycyl-aspartic acid(RGD) cell-binding domains to enhance cell adhesion.Also, hydrophilic [SQGGYGGLGSQGSGRGGLGGQT] and hydrophobic blocks [SGAGAAAAAGGAGT] were combined and cloned with different hydrophilic and hydrophobic blocks ratios. Slide13

Advantages of silk proteins as biomaterials for drug delivery:Delivery of Bioactive molecules and drugs in slow, sustained, controlled release formats. Biodegradable, biocompatible, and mechanically durable.

Processed under ambient aqueous conditions to avoid loss of bioactivity of drugs to be delivered.

Less inflammatory than other common biodegradable polymers such as poly(

lactide

) and collagen.

Proccessability

into films,

hydrogels

,

nano-fibres

, and three-dimensional scaffolds.

Degradation rate can be adjusted by controlling the crystalline state(β-sheet) during processing, in order to regulate release profile of bioactive molecules. Spider silk-based block copolymers have been designed via genetic engineering and used for the delivery of bioactive molecules, like genes and drugs. Selective delivery to target cells.Eg: Silk proteins containing tumor-homing peptides as nano-particles ---targeting tumor cells.Slide14

Preparation of

sericin

free silk solution:

Sericin

protein is a potential allergen causing allergic and

cytotoxic

reactions. Hence removal of

sericin

is necessary.

It includes the following steps as shown in the diagram:Slide15

Biosynthesis

of recombinant silk-like polymers

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APPLICATIONS OF SILK WORM SILK PROTEIN FOR DRUG DELIVERY WITH VARIOUS EXAMPLES :a. Scaffolds: Scaffolds have been prepared by using

Salt leaching method

as shown belowSlide17

b. Silk films: Prepared by cast or layer-by-layer deposition with various concentrations.Slide18

c. Nanofibres: They can be prepared by electrospinning

.Slide19

d. Microspheres: They were processed using spray drying and lipid vesicles.With spray drying microspheres of 100µm size was produced which is sub-optimal for drug delivery.Hence, lipid vesicle method is followed.Slide20

e. Microneedles: Silk fibroin based microneedles were developed for delivery of drugs and other

compounds directly to tissue in a controlled manner.

Chemical properties of the embedded substances is maintained.

Water

vapour

annealing and various temperature exposures provided control over

the diffusivity of silk

microneedles

and drug release kinetics.

a. Process of development of silk

microneedles

b. Implantation of patch of microneedles loaded with TTC Slide21

f. Nanoparticles: Silk based nanoparticles from silk fibroin solutions were stable, spherical, negatively charged, 150-170nm in average diameter and showed no toxicity.

g.

Hydrogels

:

Hydrogels

of silk fibroin are formed via sol-gel transitions by

sonication

,

vortexing

, or the presence of acid and /or ions.

h. Coatings:

Silk fibroin solution was applied as coating over the delivery systems like microspheres,

nano-particles or directly on the drug surface in order to get a sustained release of the drug. The thickness of one layer was reported to be around 10nm when deposited from a 1mg/ml silk aqueous solution. Release from these coatings can be controlled via layer thickness, number of layers and secondary structure of the fibroin layer.Slide22

Loaded bioactive moleculeType of deliveryEffect producedBone morphogenetic protein-2(BMP-2)

Scaffolds

Sustained release

Induced

Human bone marrow

stromal

cells to under go

osteogenic

differentiation

nanofibres

Supported high calcium deposition and enhanced

transcription of bone specific markers.

BMP-2, BMP-9, BMP-14MicrospheresSlow release up to 14days.BMP,RGD, Parathyroid harmone(PTH)Silk filmsDifferentiation of human bone marrow derived stem cells with silk films was induced by immobilised BMP-2Horseradish peroxidase (HRP) enzyme

ScaffoldsMicrospheresSilk filmMicroneedles

Controlled and sustained release of enzyme over 10-15days without effecting its bioactivity

Various examples of drugs that have been loaded using silk fibroin:Slide23

Loaded bioactive molecule Type of deliveryEffect produced

Adenosine

Silk films

Implants

Promote long term adenosine release

from adenosine

kinase

deficient embryonic stem cells for over a period of 2weeks via slow degradation of silk and delivery of predetermined dose

Enzymes like Glucose

oxidase

,

Lipase, HRP

Silk filmsStabilization of entrained molecules Entrained molecules retained significant activity over 10months even when stored in 37°CCurcuminNano-particles Showed higher efficiency against breast cancer cells and have potential to treat in-vivo breast tumors by local, sustained, and long-term therapeutic deliveryGrowth factorsNano-particlesMicrospheres in alginate gelsSustained release over 3weeks.More efficient in delivering BMP-2 than insulin-like growth factorsTetracyclinesMicroneedlesInhibited local infection of Staphylococcus aureusSlide24

Coatings:Eg:- 1. -Poly(lactide-co-glycolic acid) (PLGA) microspheres -Alginate microspheres Coated with silk fibroin solution formed mechanical shells as well as diffusion barrier to the encapsulated drugs.

2.Nano layer coating on small molecule drugs and therapeutically relevant proteins like

rhodamine

-B and

Azoalbumin

was achieved.

3.Multilayered silk-based coatings was given to evaluate vascular responses to heparin,

paclicoxel

, and

clopidiogrel

-----

Paclitaxel

, clopidiogrel inhibited smooth muscle cell proliferation and retarded endothelial cell proliferation.-----Silk multilayers of Heparin promoted human aortic endothelial cell proliferation while inhibited human coronary artery smooth cell proliferation which is a desired outcome in restenosis.4.Solid adenosine powder coated with silk fibroin-----showed Local and sustained delivery-----Increase in either coating thickness or crystallinity Delayed adenosine burst Decreased daily release rate of adenosine Resulting in increased duration of action Slide25

Applications of recombinant spider silk to drug delivery:

Reconstituted spider silk:

The reconstituted dragline silk proteins have been used to

prepare microcapsules for drug delivery. Microspheres may offer potential for the development of targeted drug delivery systems. Various other delivery systems into which the spider silks can be formulated have been shown in the following image.Slide26

b. Spider silk-polycation block copolymers:Poly(L-lysine) is a cationic polymer that interacts with DNA through electrostatic interactions to assemble into polyelectrolyte complexes, Which is used as an alternative to recombinant viruses for the delivery of

pDNA

into cells.

But it showed low

transfection

efficiency.

A. Schematic representation of silk-based

pDNA

complexes and silk films containing the complexes. Silk-based

polyioncomplexes

are formed between negatively charged

pDNA

and positively charged polylysine sequence of silk-polylysine block copolymer. Silk-based polyioncomplexes amd films to contain the complexes are prepared for pDNA deliveryB. pDNA complexes of the recombinant silk(yellow spots).Slide27

c. Spider silk-polycation-functional peptide

multiblock

copolymers:

Silk based block copolymers are potentially useful candidates for

nonviral

gene

vector because various functional peptides such as cell binding motifs (RGD), cell penetrating peptides(

cPP

), signal peptides of virus, and or tumor-homing peptides can be added as

ligands

through recombinant DNA techniques.

Model of receptor mediated

transfection via silk-based cationic block copolymers with ligands or functional peptides. (a) Formation of ion complexes between gene(s) and silk-polylysine block copolymers. (b) Binding of the complex to the cell via specific receptors or membrane proteins such as integrins. (c) Internalization via endocytosis and degradation of polymers in lysosomes. (d) trafficking of genes to the nucleus to initiate gene expression after the degradation of the complex. (e) Binding of adenovirus vector to the cell via the coxsackievirus and adenovirus receptor(CAR). (f) Internalization via the receptor-mediated endocytosis, involving interactions between integrins and RGDs in the adenoviral penton capsid protein. (g) Dismantling of capsid and acidification endosome, and subsequent docking at nuclear pore complexes and passage of DNA through nuclear pores via interaction of naked

capsid with microtubules and dynein motors. Slide28

Release of drug from silk matrix and fate of silk fibroin:Drug is released in a controlled manner for a long period of time.Release kinetics depends on –Adjusting

crystallinity

, concentration and structure of silk fibroin, design of delivery system as well of molecular weight and structure of embedded agents.

Eg

:

FATE:

Biosdegradation

by

proteolytic

enzymes such as

chymotrypsin

, actinase, carboxylase which involves two steps.Adsorption of silk biomaterial by different enzymesDigestion by enzymesFinal wastes are easily absorbed invivo Slide29

Conclusion

Silk-based biomaterials are used to deliver bioactive molecules, such as small drugs, proteins, genes.

They show remarkable mechanical properties, versatile processing in an aqueous environment, biocompatibility, and controlled degradation suggest silks as attractive biomaterials for controlled and sustained release, stabilization and delivery of bioactive molecules.

Silk solutions can be morphed into a variety of biomaterial formats, including films, 3D porous scaffolds,

hydrogels

, micro- and

nano

-spheres,

nanofibres

and coatings.

Targeted delivery can be achieved

Hybrid or composite silk-based materials containing other biopolymers, have not been extensively studied, yet should provide applicable mechanical, thermal, and biological properties for not only drug/gene delivery but also for tissue engineering, medical imaging, and regenerative medicine.

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REFERENCES:

1.http//www.ncbi.nlm.nih.gov/pmc/articles/PMC2658765/

2

.

http://

now.tufts.edu

/news-release/silk

microneedles

-deliver-drugs-

3.

http://en.wikipedia.org/wiki/Silk

4. Journal of control release,vol-150,issue2,10 march2011,pg no:128-1415.

5. International journal of molecular science,march-31,2009,pg no:1514-1524Slide31

QUERIES????Slide32