Patent Application
20210401934
The present invention generally relates to the field of surface functionalized nanoparticles. More specifically, the invention relates to a protein functionalized anti-inflammatory hyaluronic acid coated chitosan nanoparticle and method of preparation thereof.
Acute inflammation at a target area of a drug delivering system is a serious concern while designing nanoparticle systems which are employed in drug delivery systems Immunogenicity is an important parameter to be considered during the preparation of immune-suppressing nanoparticle systems as they are detrimental in the effective biocompatibility of the nanoparticle system. Also, uncontrolled immune stimulation, if unresolved, may lead to lethal consequences such as organ failures, which can be widespread and may further lead to mortality during conditions such as rheumatoid arthritis, stroke, hypovolemia, septic shock, sepsis, skin burns, trauma and likewise conditions.
Chitosan, a copolymer of β-(1→4)-linked D-glucose-2-amine and N-acetyl-D-glucose-2-amine, has been exhaustively employed in drug delivery systems. However, a cationic surface associated with chitosan nanoparticles, reduces the circulation time and bioavailability of the nanoparticle system in a biological environment. Now, while chitosan nanoparticles further associated with anionic polysaccharides, increase circulation time, rate of macrophage uptake is decreased. Therefore, chitosan nanoparticles associated with anionic polysaccharides require an association for further triggering phagocytosis.
Therefore, there exists a need for a biocompatible nanoparticle system with immune-suppressing properties to prevent/reduce acute inflammation and increase the blood circulation time.
The accompanying figures where like reference numerals refer to identical or functionally similar elements throughout the separate views and which together with the detailed description below are incorporated in and form part of the specification, serve to further illustrate various embodiments and to explain various principles and advantages all in accordance with the invention.
Before describing in detail embodiments that are in accordance with the invention, it should be observed that the embodiments reside primarily in a protein functionalized anti-inflammatory hyaluronic acid coated chitosan nanoparticle (HA-CS NPs) and a method of preparation thereof. The method of preparation includes functionalizing of the HA-CS NP with surface adsorbing anti-inflammatory proteins.
In this document, the terms “comprises,” “comprising,” or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article or composition that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article or composition. An element proceeded by “comprises . . . a” does not, without more constraints, preclude the existence of additional identical elements in the process, method, article or composition that comprises the element.
Various embodiments of the invention provide a protein functionalized anti-inflammatory HA-CS NP and a method of preparation thereof.
In accordance with the present invention, a protein functionalized anti-inflammatory HA-CS NP includes a central core and an outer shell surrounding the central core. The central core in accordance of the invention includes the HA-CS NP and the outer shell comprises functionalized surface adsorbed anti-inflammatory proteins, bound to the central core. The anti-inflammatory proteins functionalized to the HA-CS NP are ITIH4 (inter-alpha-trypsin inhibitor heavy chain 4) and AGP (alpha-1-acid glycoprotein, also known as orosomucoid). The central core and the functionalized anti-inflammatory particles undergo a chemisorption process, thereby the ITIH4 protein forming an ester bond between the carboxyl groups of the aspartates at their C′ termini and the C-6 hydroxyl groups of the HA. Furthermore, the AGP protein chemisorbed onto the HA layer forms a glycosidic bond through the glycan branches of AGP.
In one embodiment, a combination of anti-inflammatory proteins ITIH4 and AGP are bound to the central core HA-CS NP, forming stabilized nanoparticle systems by virtue of the strong covalent bonds formed between the anti-inflammatory proteins rendering a stable protein corona.
In another embodiment, the anti-inflammatory proteins bound to the central core may include either ITIH4 or AGP.
Referring to Table 1, Table 1 depicts the list of adsorbed proteins forming the protein coronas of the by CS NPs, HA-CS NPs, and Alg-CS NPs respectively, thereby listing the adsorbed proteins.
Hereinafter, a method of preparing the protein functionalized anti-inflammatory HA-CS NP is described.
The method of preparation of the protein functionalized anti-inflammatory HA-CS NP begins with dispersing chitosan nanoparticles in 0.1 M acetic acid/acetate buffer at a pH of 5, to produce a dispersion. The ensuing step includes adding an equal amount of acetate buffer containing hyaluronic acid at a concentration of 1.5 mg/ml, under vigorous stirring for a time period of 30 minutes at 1200 rpm to form HA-CS, wherein the HA-CS NP is dialyzed against deionized water. Thereafter, in a crucial step, the HA-CS NP undergoes functionalizing with surface adsorbing anti-inflammatory proteins as illustrated in
Functionalizing of the HA-CS NPs with surface adsorbing anti-inflammatory proteins includes a plurality of steps. More specifically, the functionalizing of the HA-CS NPs with surface adsorbing anti-inflammatory protein Alpha-1 Acid glycoprotein (AGP) includes dissolving the Alpha-1 Acid glycoprotein (AGP) in PBS buffer at a pH of 7.4, at a first step. In a next step, the dissolved Alpha-1 Acid glycoprotein (AGP) is mixed with 1 ml of HA-CS NPs at 0.25 mg/mL to reach a concentration of 100 μg/mL of AGP functionalized HA-CS NPs (AGP-HA-CS NP). Subsequently, HA-CS NPs in the presence of varying doses of AGP including 5, 10, 20, and 30 μg/ml were gently mixed to form varying suspensions, followed by vortexing for a time duration of 1 minute to obtain four homogenous suspensions of AGP-HA-CS NP, as illustrated in
The method of preparing CS NPs further includes numerous steps. A first step of preparation of chitosan particle includes dissolving chitosan at a concentration of 0.07% weight, in 4.6 mM of HCl. The pH of the chitosan solution was then adjusted to 5 by adding appropriate volumes of 0.1 M NaOH and further underwent continuous stirring overnight. In the ensuing step, 0.1% weight, Triphenyl phosphate solution (TPP) in deionized water was prepared and the pH of the solution was adjusted to 5 using 0.1 M HCl. The prepared TPP solution was added to the chitosan solution in a mass ration of 1:9, to prepare a complexation solution. Considering an example, 0.214 mL of TPP solution was added to 2.786 mL of CS solution, where the final concentrations of TPP and CS represented 0.0071 and 0.064% weight respectively, thereby resulting in a 1:9 mass ratio of TPP:CS. All solutions were filtered through a 0.22 μm pore size filter. The complexation solution further undergoes magnetic stirring and agitation, for a time period of 30 min at room temperature. Finally, the complexation solution is left undisturbed for a period of 24 hours after a brief sonication to produce CS NPs. The dispersed CS NPs were then dialyzed against deionized water (MWCO 1000 kDa).
Characterization Studies:
As illustrated in
Further, moving to
In an embodiment, the protein functionalized anti-inflammatory HA-CS NPs, specifically AGP-HA-CS NPs is employed in suppressing the immunity of activated cancer cells. AGP-HA-CS NPs is added to activated breast cancer cells (MDA-MB-231), with activated migration by virtue of LPS (Lipopolysaccharide). LPS (Lipopolysaccharides) enables accelerated migration of cancer cells, thereby acting as a stimulated immune response. The activated breast cancer cells are grown to near confluency and further scratched to create a wound.
In another embodiment, the protein functionalized anti-inflammatory HA-CS NP with surface adsorbed proteins AGP is employed in a drug delivery system. Considering an example, a widely known immune suppressant corticosteroid, namely dexamethasone (DXM) is delivered by the protein functionalized HA-CS NP in accordance with the present invention to reduce acute inflammatory responses in conditions like rheumatoid arthritis, stroke, hypovolemia, septic shock, sepsis, skin burns, trauma and likewise.
Those skilled in the art will realize that the above recognized advantages and other advantages described herein are merely exemplary and are not meant to be a complete rendering of all of the advantages of the various embodiments of the invention.
In the foregoing specification, specific embodiments of the invention have been described. However, one of ordinary skill in the art appreciates that various modifications and changes can be made without departing from the scope of the invention as set forth in the claims below. Accordingly, the specification and figures are to be regarded in an illustrative rather than a restrictive sense, and all such modifications are intended to be included within the scope of the invention. The benefits, advantages, solutions to problems, and any element(s) that may cause any benefit, advantage, or solution to occur or become more pronounced are not to be construed as a critical, required, or essential features or elements of any or all the claims. The invention is defined solely by the appended claims including any amendments made during the pendency of this application and all equivalents of those claims as issued.
| Number | Date | Country | Kind |
|---|---|---|---|
| 117390079 | Oct 2017 | SA | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/SA2018/000011 | 4/9/2018 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62483515 | Apr 2017 | US |
