Berberine two different materials with wide range

       Berberine has long been used as a
traditional medicine because of  its
potential activity  against Bacterial,
fungal, viral diseases. Berberine have a 
complex and diverse chemical structure provide a base for different
biological targets. The present study deals with the novel encapsulation of
biopolymer called Collagen1 with berberine by Electrospinning method  in different compositions, parameters, and
method for preparation. By electrospinning not only  we get desired mechanical and biological
properties of encapsulation studies, but also used to combine these two
different materials with wide range of drug delivery science and tissue
engineering  properties to produce
electrospun nanofibres at different compositions. The physical and thermal
properties of electro spun nanofibres interaction and  were examines under FE-SEM. resultant
studies  may be used in variety of
applications including Chemical, Genetical, Immunological  and industrial purposes

 

Introduction

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        Encapsulation studies were mainly
useful for tissue engineering and meeting a wide range of applications in
Biotechnology, Environmental, and Medicine fields. Mainly these studies made
possible in nano particles engineering, which enhances the site  specific drug delivery system and improves
the pharmaco kinetics properties to drug. There are many methods to perform
encapsulation studies, in this study we discuss abut electrospinning method.
Electro spinning is a method which uses electrical energy as a source  and fabricate fibres in different diameter
ranges from  microns to nano meters.

        Berberine is a plant alkaloid with many
biological activities. Preclinical in vitro and in vivo studies
carry diverse pharmacological actions of berberine that could be potentially
helpful in the management of infectious, anti-bacterial anti-inflamatory and metabolic
diseases. To study more about we conducted the study in nanotechnology by
infusing berberine in collagen for drug delivery experiments for that we will
get to know about encapsulation  nature of
drug. The purpose of this study is to identify the potential of drug by using
electrospinning at  different
compositions  of berberine infused
collagen.

 

 

Materials
and Methods 

 

Collagen cross
linking:

 

The collagen 1
was cross-linked by dealing with glutaraldehyde vapor, soaked with 20%
glutaraldehyde solution at room temperature for different time period, followed
by treatment with 0.2M glycine aqueous solution to lump un reacted Amino acids.

 

Preparation
of solutions:

 

collagen
type I is extracted  from rat tail calf
skin, the proteins were dissolved in HCL for 8 hours at room temperature.
Whereas the berberine solution was prepared by dissolving  50 mg of berberine in 2ml of methanol and
then the emulsify it with 1% Pva solution and run in  cyclo mixture for 2 hours to homogenise the
solution. The mixture of   berberine and
collagen solutions were prepared in the weight ratio of 1:2, 1:1, 2:1 for
encapsulation

 

 

Parametres:

  

The
electrospinnig instrument can be used for various solutions, but every solution
has its own properties. To prepare  the
encapsulation mainly we need ,Drum rotation speed, Syringe translation speed,
Solution flow rate, Voltage required the following table has listed the parameters
we used for encapsulation Preliminary experiments revealed that, independent of
the conditions used, continuous fibres could not be spun from acidic aqueous
solutions of pure collagen. It is known that by addition of sodium chloride to
the solution, formation of fibres by electrospinning becomes possible due to
the increase in solution conductivity . Moreover, the presence of NaCl can
induce hydrophobic interactions in or between the protein molecules and thus
contribute to the production of continuous fibres The higher net charge
increases the force exerted on the jet and at a concentration of 42.5mM NaCl
and spinning voltages between 10 and 25 kV, fibre formation was observed.
However, continuous jets could not be produced and only short beaded fibres
were obtained. This latter phenomenon is known to be favoured by the presence
of a high electric field, which leads to capillary breakup of the
electrospinning jet

 

Increasing
the viscosity of the spinning solution is a way to overcome the formation of
beads and a suitable polymer for this purpose we use PVA. Addition of this
polymer to the protein solution, which also contained 42.5mM NaCl, allowed a
much better control over fibre formation. The voltages necessary to obtain a
continuous fibre were dependent on the weight ratio of collagen to Berberine.
Collagen and berberine solutions (2% w/v) containing 42.5mM sodium chloride,
having a collagen and berberine weight ratio of 1:2 and spun at 21 kV, afforded
the formation of a continuous jet. However, the collected fibres were not
completely dry and resulted in meshes of highly fused fibres. Increasing the
weight ratio between collagen and Berberine to 1:1 or 2:1 required higher
potentials of 22 and 23.5 kV, respectively. However, at a collagen and berberine
weight ratio of 1:2, beaded and highly fused fibres were obtained. Obviously,
under these conditions the water evaporation from the fibres was not complete.
Dry fibres entirely devoid of beads and with a narrow diameter distribution
(average fibre diameter ¼ 0.4070.05 mm) were produced at a weight ratio of
collagen and berberine equal to 1:1.

 

Electrospinning:

The
electrospinning method consists a syringe needle, electrode, stainless sheet
paper on drum and electric supply. 5ml syringe filled with polymer  solution which is linked to syringe pump. Solutions
will be hard pressed through tube on a rotating drum which is covered by stainless
steel paper. The  needle( distance
between needle and rotate drum should be 6cm to collect the fibres) was
associated to high voltage supply (Maximum volts 40 kv and the experiment has
to be carried at room temperature ). The solution will get a positive voltage
which is 30 kv and the fibres were collected on steel paper with the speed of
0.5 ml/hour

 

SEM

 

The resultant
Microscopic images of different composition of polymer encapsulation has viewed
under FESEM-EXT-501 microscope.

 

Results

 

 

Different
compositions of Berberien and collagen mixtures(2:2, 1:1, 1:2)  were performed to produce fibres from soluble
berberine. As with collagen solutions, addition of NaCl at a concentration of
42.5mM and PVA at a concentration of 1% w/v were necessary to obtain a
continuous fibres., it was possible to spin fibres at a voltage of 10 kV and a
flow rate of 50 ml/ min. These fibres had an average thickness of 0.5 mm, The
fibres have a rough surface and appear to be composed of 5–10nm wide filaments,
oriented parallel to their longitudinal axis similarly to native elastin fibres
As with collagen, the Berberine appear to preserve the ability to self organize
into the native structure during fibre formation in the electrospinning
process. The fibres were easily produced, but difficult to collect because of
substantial splaying. Splaying occurs when the radial forces derived from the
electrical charges carried by the jet, overcome the cohesive forces in the jet
itself. The single jet divides into many charged jets before reaching the
collecting plate. Splaying thus yields unusual small diameter fibres In the
arterial wall, collagen and elastin are both present and constitute together
with the extracellular matrix and the cells, a fibre-reinforced composite
structure of which the mechanical properties are mainly determined by the
fibrous network. The presence of both proteins is necessary to confer the
vessel its strength but also its elasticity

 

In
the arterial wall, collagen and Berberine are both present and constitute
together with the extracellular matrix and the cells, a fibre-reinforced
composite structure of which the mechanical properties are mainly determined by
the fibrous network. The presence of Both mixture concentrations is necessary
to confer the vessel its strength but also its elasticity  Aqueous solutions comprising collagen,
Berberine (weight ratios are, 2:1, 1:1, 1:2) and

 

The
degree of cross linking was estimated by determining both the denaturation
temperature and the residual amount of free amine groups of (non-)cross linked
samples. Formation of crosslinks in the collagen/ Berberine spun matrices
increased the denaturation temperature of the samples. As a consequence of
crosslinking, the amount of free amino groups present in the samples decreased
Independent of the weight ratio of collagen to Berberine, the relative
percentage of free amino groups left after cross linking of the fibres was
decreased to approximately 30% of the original value. These results are
comparable with those obtained by performing the same reaction with
freeze-dried scaffolds of insoluble collagen and elastin In the DSC thermograms
of dry uncrosslinked fibres. Moreover, by means of SEM, it was verified that no
NaCl crystals were present at the surface of the fibres after EDC/NHS
crosslinking.. Crosslinked collagen/berberine scaffolds with different weight
ratios (2:1, 1:1, 1:2, ) of the two solutions were formed. In all cases, the
formation of a confluent multi-layer of SMC, growing on top of each other was
observed by means of histology The possibility to electrospin collagen and
berberine solutions into fibres composed of a homogeneous blend of the two
solutions can lead to the production of scaffolds with extraordinary
properties, completely different from those observed in analogous mixtures of
Solutions , for which the separate contributions can always be well
distinguished. Evaluation of the specific interactions occurring in or between
soluble collagen and soluble berberine and theoretically resulting in formation
of collagenous fibrillar structures and aggregation of collagen and berberine might
result in a better understanding of the potential of the application of
collagen/berberine electrospun scaffolds in different fields, like tissue
engineering. Further investigations are actually being performed in this
direction.

 

Conclusion:

 

Electrospinning
was used as a successful technique to produce non-woven meshes from aqueous
solutions of collagen type I and Berberine. In all cases, the addition of NaCl
was necessary to spin homogeneous and continuous fibres. Composition of the
solution, net charge density and applied electric field were parameters
influencing the morphology of the obtained fibres. Spinning collagen/elastin
solutions yielded meshes composed of fibres with diameters ranging from 220 to
600nm. Stable scaffolds were prepared by cross linking with EDC/NHS. After
cross linking, scaffolds completely devoid of NaCl were obtained. SMCs were
successfully cultured on cross linked scaffolds and a confluent layer of cells
was observed after 14 days on the surface of the different meshes. One of the
advantages of electrospinning aqueous solution of collagen and Berberine is the
formation of scaffolds with high porosity and surface area, two essential
requisites for tissue engineering. Electrospinning solutions of the two
solutions separately from each other can also give the possibility to produce
multilayered scaffolds with controlled morphology and/or mechanical properties.
Moreover, in this study, it has been shown that fibres, in which the two solutions
cannot be distinguished, can be electrospun from a mixture of collagen and
Berberine. This may result in fibres with extraordinary mechanical properties,
different from those observed in analogous mixtures of the insoluble solutions.
Further investigations are currently being done in this direction.

 

Acknowledgement:

 

 I wish my sincere thanks towards the SRM
University, Chennai for providing us the SEM Images