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Vitamin B12 grafted Layer-by-Layer liposomes bearing HBsAg facilitates oral immunization: Effect of modulated biomechanical properties Ashwni Kumar Verma, Shweta Sharma, PRAMOD KUMAR GUPTA, Deepak Singodia , Shaswat Kansal, Veena Sharma, and Prabhat Ranjan Mishra Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00274 • Publication Date (Web): 24 May 2016 Downloaded from http://pubs.acs.org on May 27, 2016
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Molecular Pharmaceutics
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Vitamin B12 grafted Layer-by-Layer liposomes bearing HBsAg facilitates oral immunization:
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Effect of modulated biomechanical properties
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Ashwni Kumar Verma1, Shweta Sharma1, Pramod Gupta1, Deepak Singodia1, Shaswat Kansal1, Veena
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Sharma2 and Prabhat Ranjan Mishra1*
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1- Division of Pharmaceutics, CSIR-CDRI, Lucknow, INDIA
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2- BioScience & Biotechnology Division, Banasthali Vidyapith, Rajasthan, INDIA
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Email:
[email protected];
[email protected] 8 9 10 11 12 13
*Corresponding Author
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Dr. P. R. Mishra Ph. D
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Division of Pharmaceutics,
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Preclinical South PCS 002/011,
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CSIR-Central Drug Research Institute
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B.S. 10/1, Sector-10, Jankipuram Extension,
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Sitapur Road, Lucknow-226031,
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India
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Molecular Pharmaceutics
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Abstract:
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Adhesion forces of nanoparticulate materials towards biological membrane are crucial for designing
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delivery system for therapeutic molecule and vaccines. Present study aims to investigate the impact of
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surface roughness of nanoparticulate system in oral delivery of antigen and its targeting to towards
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intestinal APCs. To evaluate this hypothesis Layer by layer coated liposomes (LBL-Lipo) were
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fabricated using sodium alginate and Vitamin B12 conjugated Chitosan (VitB12-Chi) as anionic and
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cationic polyelectrolyte respectively. Change in surface roughness was observed on changes in pH from
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gastric to intestinal conditions attributed to increase and decrease in charge density on VitB12-Chi.
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Surface roughness was measured in terms of RMS measured by topographical analysis using AFM.
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LBL-Lipo were further characterized for their size, zeta potential and release behavior to evaluate the
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potential for oral vaccine delivery. In vitro cell uptake in macrophage cells (J-744) shows about 2 and
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3.1 fold increased uptake of rough LBL-Lipo over smooth LBL-Lipo at 37°C (endocytosis) and 4°C
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(endocytosis inhibition) indicating improved biological interaction. Further in vivo immunization study
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revealed prototype formulation were able to produce 4.8 and 3.3 fold higher IgG and IgA levels in
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serum and feces respectively in comparison to smooth LBL-Lipo.
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Key words: AFM, Biomechanical properties, Mucus penetration, peyer's Patches, Vitamin B12, and
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APCs
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Molecular Pharmaceutics
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Introduction:
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Oral delivery has always been most preferred route for administration of therapeutics including vaccines
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or drugs and this is because of its convenience in administration which leads to improved patient
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compliance. In case of vaccine delivery oral route plays an important role because oral administration of
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antigens induces both mucosal as well as systemic humoral and cellular immune response 1. Although,
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oral delivery of vaccines or antigenic proteins possesses several advantages, yet there delivery remains a
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major challenge because of the rapid degradation under the adverse gastric conditions. Encapsulation of
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antigen (Ag) in microparticles or nanoparticles is one of the approaches that has been extensively
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studied for mucosal delivery systems of antigen2. The particulate carrier can not only protect the Ag
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through enzymatic attack but can also be made to transport antigen directly to Peyer’s patches via M –
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cells with precise modification. Peyer’s patches are specialized structures which are basically composed
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of M cells and actively participates with underlying immune cells (Antigen presenting cells) in
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providing immunity against inhaled pathogens by providing systemic antibodies as we as mucosal
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Antibodies (IgA) that acts at multiple sites like nasal, oral and vaginal mucosa3. Several reports are
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available in literature where different types of particles have been developed for delivery of vaccines
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and states that particles with mucoadhesive nature and size < 1 µm can effectively be taken up by M
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cells 4.
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Another aspect that has been focus of modern medicine is the alteration in surface geometry of
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particulate carriers for enhanced delivery of therapeutic molecule to target cell. Recently many groups
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are working on the concept that, alteration in surface geometry of particles can alter their interaction
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with biological system. Surface roughness is one such parameter that comes under the surface geometry
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and several studies have suggested that improved surface roughness enhance biological interaction
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leading to improved uptake in target cell 5. However, no such report is available where this concept has
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been applied for oral delivery of vaccines.
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In this study, we report the development of layer by layer coated Liposomes (LBL-Lipo) with rough
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surface for oral delivery of Hepatitis B surface antigen (HBsAg). Layer by layer coating (LBL) with
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oppositely charged polyelectrolytes was performed over liposomes because liposomes itself are unstable
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systems which otherwise degrades in gastric conditions due to the presence of bile salts and pancreatin 6.
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LBL coating makes the liposomes
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advantages over other lipidic and polymeric nanoparticles (viz., their flexible shape and improved
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responsive release).7 Layer-by-layer coating of nanoparticles provides better protection to encapsulated
robust for oral delivery and also provides several additional
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Ag against gastric enzymes8, 9. Further in the present study Vitamin B12 conjugated chitosan (VitB12Chi) was selected as positively charged polyelectrolyte for the development of rough surface “LBLLipo” because of several reasons. First, it is non–toxic, biocompatible and biodegradable. Second unlike chitosan it is water soluble at all GIT conditions. Third and most important is that it participates in pH induced rearrangement of multiple layers of liposomes resulting in generation of particles with rough surface. Basically, in the present study we have conceptualized to develop pH induced rearrangement in the layers of “LBL- Lipo” which presents rough surface particles at the target site in in-vivo conditions and this has been done by means of VitB12-Chi conjugate. VitB12 has a pKa 2.0, due to which it possess net increase in positive charge at gastric pH 1.5 in comparison to duodenum pH 6.5. The present study investigates that how the change in pH of GIT conditions leads to alteration or shift in zeta potential or charge which facilitates increase in surface roughness of final LBL-Lipo available at target site. Further, by means of Atomic force microscopy (AFM) we have tried to establish our proof of concept that how this increase in surface roughness of LBL-Lipo improves adhesion towards lipid bilayer leading to improve bio-interactions.
Experimental Section Materials Chitosan with a degree of N-deacetylation of 75−85% (65–90 kDa), Sodium alginate (15−20 cP, 1% in H2O),
N-hydroxysuccinimide
(NHS),
Vitamin
B12,
dimethylaminopropyl) carbodiimide hydrochloride (EDC),
Succinic
anhydride,
1-ethyl-3-(3-
of 4-Dimethylaminopyridine (DMAP) ,
Soya phosphatidylcholine (from soybean, Type IV-S), cholesterol, Succinic acid, trifluoroacetic acid (TFA), flourescein isothiocyanate (FITC), Pluronic F68, sephadex G100, Sulfo-rhodamine , Bradford reagent, LDH Assay kit and Bovine serum albumin were purchased from Sigma Aldrich (USA). HBsAg, Anti HBsAg Abs, anti-B220-FITC and Secondary Abs were purchased from Santacruz biotechnology (USA). Analytical grade sodium chloride, sodium phosphate dibasic anhydrous, sodium bicarbonate, methylene chloride and glacial acetic acid, were purchased from SD Fine Chem India. LysoTracker® Deep Red was purchased from Thermo Fischer Scientific (U.S.A).
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Methods Synthesis and characterization of VitB12-Chi VitB12-Chi conjugate was synthesized using carbodiimide chemistry. In brief, first of all VitB12 was succinylated in the presence of DMAP under nitrogen atmosphere. This succinylated VitB12 was then conjugated to chitosan in the presence of EDC and NHS by means of carbodiimide chemistry. Free reactants and byproducts were removed by dialyzing the product in Milli-Q water for 48 hrs. Finally the conjugate was lyophilized to obtain the dry product. The conjugate was characterized by proton NMR. The proton NMR was performed in D2O. The degree of substitution (DS) was determined by TNBS (2,4,6-Trinitrobenzenesulfonic acid ) assay. For TNBS assay freeze dried VitB12-Chi conjugate was dissolved in milli Q water, VitB12-Chi concentrations (10, 20, and 30µg/ml) were incubated with 2mL of 4% (w/v) NaHCO3 and 0.1% (v/v) TNBS reagent at 37°C for 2 hrs. Then, 2mL of 2 M HCl was added and the absorbance was recorded at 344 nm using UV-Vis Spectrophotometer (UV -1700, Shimadzu, Japan). Final DS was calculated using the standard curve of chitosan 10.
Preparation of HBsAg loaded liposomes HBsAg loaded plain liposomes were prepared by thin film hydration method11. In brief L-αPhosphatidylcholine (Type IV-S > 30 %) and cholesterol in 7:3 Mole ratio were dissolved in chloroform (2 ml) and evaporated using rotavapor to form thin film at 45°C. This thin film then rehydrated in Phosphate buffer saline (PBS pH 7.4) containing HBsAg and subsequently sonicated using probe sonicator and extruded from 200 and 100 nm pore size polycarbonate filters to get unilamellar vesicles (ULVs) from multi lamellar vesicles (MLVs). Different variable were optimized to obtain the liposomes of size 30 %) and cholesterol in 7:3 Molar ratio and then chloroform was evaporated using rota vapor to form thin film at 45°C. Further rehydration of the film and LBL coating was done similar to HBsAg loaded LBL-Lipo.
Immunization studies All the protocols for animal studies were approved by the Institutional Animal Ethical Committee of CSIR-CDRI Lucknow (INDIA). In-vivo immunization studies were performed in 3–4 weeks old male BALB/c mice. Just like the previous in vivo targeting study immunization study were also performed with gastric alkalinization because of the similar reason. Here he mice were divided into 6 groups of 8 animals each. The first four groups received free HBsAg, HBsAg loaded Plain Lipo, smooth LBL-Lipo and Rough LBL-Lipo at equivalent dose of 5µg/mice orally along with Gastric alkalinizing agent. The fifth group received HBsAg loaded LBL-Lipo (prototype formulation) orally in PBS (pH 7.4) without 10 ACS Paragon Plus Environment
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Molecular Pharmaceutics
gastric alkanizing agent and is expected to change its surface roughness while passing from gastric to intestinal conditions. Remaining sixth group received free HBsAg in PBS via intra muscular (IM) route at equivalent dose of 2µg/mice. All groups received one vaccination dose at initial day and a booster dose after one week of initial dose. Blood samples were taken on day 0 (prior to the vaccination), at day 7 (prior to boosting) and subsequently at 2, 3, 4, 6, 8 and 12 week after initial dosing. The samples were centrifuged at 1000 g for 10 min to collect the serum and stored at −80 °C until analysis for Ab titers. Feces of each mouse group were collected 1, 2, 4, 6 and 14 days after booster dose. Five fecal pellets were selected randomly and IgA were isolated19. Fecal pellets were incubated for 15 min in homogenization buffer (50 mm EDTA, 1% BSA, 0.1 mg/ml soybean trypsin inhibitor and 1 mM AEBSF in PBS, pH 7.4, 20 µl buffer per mg feces) on ice, followed by mashing it with a blunt needle. Samples were centrifuged and stored at -80°C.
Antibody titers Samples from all time points were analyzed by performing ELISA, 50 µl of HBsAg (1µg/ml) in PBS was added to 96-well ELISA plates and incubated overnight at 4°C. Plates were blocked with 50 µl of blocking buffer (5% BSA in PBS) for 1 h at 25°C 50µl of diluted serum samples were added in each well and incubated for 2 hrs at 25°C. Plate was washed twice with wash buffer (0.2% v/v Tween 20, 0.5% w/v BSA in PBS) and treated with 50µl of anti mice IgG/ IgA secondary Abs (Santa cruiz Biotechnology) and incubated for 2 hrs. Finally, plates were washed and developed with TMB substrate. The reaction was stopped by adding 20µl of 1N HCl and absorbance was taken at 450 nm. Titer was calculated as the inverse dilution at which the sample matched that of the average absorbance from standard samples.
Statistical analysis Statistical significance among different groups was calculated using student t-test. Difference was considered significant when PIgG1>IgG2a) (Fig. 9 B) 41.
IM-HBsAg vaccinated mice did not show any detectable amount of HBsAg specific IgA Abs in feces that supports that they do not provide mucosal immunity due to their root of administration
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. In
contrast to I.M administered HBsAg, orally administered LBL-Lipo (prototype formulation), Rough LBL-Lipo and Smooth LBL-Lipo showed detectable IgA Abs levels in feces after two weeks of administration, indicating the advantage of oral delivery of vaccine over I.M delivery. The levels of IgA 26 ACS Paragon Plus Environment
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Molecular Pharmaceutics
Abs were found to increase sharply from 2nd week and reached maximum at 6th week. Also the IgA Abs levels observed after 6th week of administration were found to be 3.3 fold higher in both LBL-Lipo (Prototype formulation) (1593.1±345.1mIU/gm of feces) and Rough LBL-Lipo (1614.2±224.51mIU /gm of feces) in comparison to smooth LBL-Lipo (479.0±193.4mIU/gm of feces) (Fig 9 C).Similar to IgG Abs response orally administered free HBsAg and -HBsAg loaded plain liposomes did not show any detectable amount of HBsAg specific IgA Ab. The minimal difference obtained in antibody response from rough LBL-Lipo with alkanizing agent and LBL-Lipo (prototype formulation) without alkanizing agent indicates that our formulation behaves in similar pattern as expected and improves the surface roughness at the target site in intestine. Further the enhanced antibody response with rough LBL-Lipo than smooth LBL-Lipo confirms that the positive response is due to generation of rough surface which improves the interaction of LBL-Lipo with APCs and thereby improves the immune response. Finally this enhanced performance obtained with rough LBL-Lipo can be explained by the contribution of several factors. First, is the enhance protection of entrapped antigen in LBL-Lipo which protect its degradation from proteolytic enzymes, Second is the improved mucus penetration and retention of rough LBL-Lipo and third is the enhanced antigen presentation towards intestinal APCs by LBL-Lipo which is assisted by improved endocytic uptake by APCs.
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30000
A
HBsAg IM HBsAg (Oral) LBL Lipo (Oral) Rough LBL Lipo Smooth LBL Lipo Lipo
25000 20000
** **
15000
**
**
**
10000 5000
12000
0
1
+
2
B
3 4 Time (weeks) IgG1 IgG2a IgG2b IgG3
9000 6000 3000 0
o M) r a l) lip o -lip g (I BL BL o (O L p L sA i l h h B LH ug oot LB Ro Sm
L ip
IgA titers (mIU/gm feces)
0
Ab titer mIU/ml
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Serum IgG titer (mIU/ml)
Molecular Pharmaceutics
6
8
12
2500
C
LBL Lipo (Oral) Rough LBL Lipo Smooth LBL Lipo
2000
** **
1500 *
1000
o
500 0
1
+
2
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Time (week)
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Fig. 9: Antibody titers for oral and I.M. vaccinated mice with HBsAg and HBsAg –LBL-Lipo formulations (5µg/animal oral and 2µg/animal I.M.). (A) Serum profile of Total IgG antibodies against HBsAg after Oral and IM vaccinated mice with booster dose after one week of initial dosing. (B) Serum IgG subtype titer after 4 week of initial dose. (C) Mucosal IgA antibodies against HBsAg after oral vaccination with HBsAg LBL-Lipo (200µg/Kg) and Booster of equal dose one week of initial dosing. (Other groups (HBsAg-IM, HBsAg-Oral and HBsAg-Lipo) did not produce detectable amount of secretary mucosal IgA) (n=5).
+ - Booster dose, (**) - significant difference at P
