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14 Δεκ 2012 (πριν από 4 χρόνια και 4 μήνες)

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SILK
-
BASED DELIVERY SYSTEMS
OF BIOACTIVE MOLECULES

Presented by:



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What is a silk based delivery
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CONTENTS


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
.














Beta pleated structure of the silk

Amino acid composition of the
fibroin


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 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

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

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
-
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.




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.

Loaded bioactive
molecule

Type of delivery

Effect produced

Bone morphogenetic
protein
-
2(BMP
-
2)

Scaffolds



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nanofibres

Supported high calcium deposition and
enhanced

transcription of bone specific
markers.

BMP
-
2, BMP
-
9, BMP
-
14

Microspheres

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

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

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


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s瑯牥搠楮i

°
C

Curcumin

Nano
-
particles

Showed higher efficiency

against breast
cancer cells and have potential to treat in
-
vivo breast tumors by local, sustained, and
long
-
term therapeutic delivery

Growth

factors

Nano
-
particles

Microspheres in
alginate gels


Sustained release over 3weeks.


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-
㈠瑨慮t
insulin
-
like growth factors

Tetra
cyclines

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 th
e
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
-
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
-
1524

QUERIES????