Transcript 1449199_634756089580756072x

SILK-BASED DELIVERY SYSTEMS
OF BIOACTIVE MOLECULES
Presented by:
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What is a silk based delivery??
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CONTENTS
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Introduction
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.
 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
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.
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.
a. 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.
Amino acid composition of the
fibroin
Beta pleated structure of the silk
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
highly conserved sequences consisting
Of short side-chain amino acids such as
Glycine and alanine.
Small hydrophilic blocks with more
complex sequences that consist
aminoacids with bulkier side-chains
and charged aminoacids.
Show α-helices in the solution
And β-sheet structures in the
assembled form
Thus 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.
a. Secretion of silk thread from the spinning duct
b. Structure of spider silk
fibroin
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
protein
Releases protons
in aqueous
solution,
resulting in pH-4
Potassium nitrate
Prevent from
denaturing in
the acidic
medium
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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
fibroins
Called as
fibroins or
spidroins
specifically
and contains
light and heavy
counter-parts
Mechanical
properties
Weaker and
less extensible
Stronger with
high extensible
properties
Spinning
conditions
Either strong
or elastic
Both strong
and elastic
2.Recombinant silk proteins:
a. Silk worm variants:
 Silk like repeats of (GAGAGS), elastic block copolymers, Silk-elastinlike 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, celladhesive 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.
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.
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:
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 below
b. Silk films:
Prepared by cast or layer-by-layer deposition with various
concentrations.
c. Nanofibres:
They can be prepared by electrospinning.
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.
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
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.
Various examples of drugs that have been loaded using silk
fibroin:
Loaded bioactive
molecule
Type of delivery
Effect produced
Bone 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- Microspheres
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Slow release up to 14days.
BMP,RGD,
Parathyroid
harmone(PTH)
Silk films
Differentiation of human bone marrow
derived stem cells with silk films was
induced by immobilised BMP-2
Horseradish
peroxidase (HRP)
enzyme
Scaffolds
Microspheres
Silk film
Microneedles
Controlled and sustained release
of enzyme over 10-15days without
effecting its bioactivity
Loaded
bioactive
molecule
Type of
delivery
Effect 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 films
•Stabilization of entrained molecules
•Entrained molecules retained significant
activity over 10months even when stored in
37°C
Curcumin
Nano-particles
Showed higher efficiency against breast
cancer cells and have potential to treat invivo breast tumors by local, sustained, and
long-term therapeutic delivery
Growth factors
Nano-particles
Microspheres in
alginate gels
•Sustained release over 3weeks.
•More efficient in delivering BMP-2 than
insulin-like growth factors
Tetracyclines
Microneedles
Inhibited local infection of
Staphylococcus aureus
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
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 delivery
B. pDNA complexes of the recombinant silk(yellow spots).
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.
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 enzymes
Digestion by enzymes
Final wastes are easily absorbed invivo
REFERENCES:
1.http//www.ncbi.nlm.nih.gov/pmc/articles/PMC2658765/
2.http://now.tufts.edu/news-release/silk microneedles-deliverdrugs3. 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:1514-1524
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