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Novel Gemcitabine Conjugated Albumin Nanoparticles: a Potential Strategy to Enhance Drug Efficacy in Pancreatic Cancer Treatment

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Abstract

Purpose

The present study reports a novel conjugate of gemcitabine (GEM) with bovine serum albumin (BSA) and thereof nanoparticles (GEM-BSA NPs) to potentiate the therapeutic efficacy by altering physicochemical properties, improving cellular uptake and stability of GEM.

Methods

The synthesized GEM-BSA conjugate was extensively characterized by NMR, FTIR, MALDI-TOF and elemental analysis. Conjugation mediated changes in structural conformation and physicochemical properties were analysed by fluorescence, Raman and CD spectroscopy, DSC and contact angle analysis. Further, BSA nanoparticles were developed from BSA-GEM conjugate and extensively evaluated against in-vitro pancreatic cancer cell lines to explore cellular uptake pathways and therapeutic efficacy.

Results

Various characterization techniques confirmed covalent conjugation of GEM with BSA. GEM-BSA conjugate was then transformed into NPs via high pressure homogenization technique with particle size 147.2 ± 7.3, PDI 0.16 ± 0.06 and ZP -19.2 ± 1.4. The morphological analysis by SEM and AFM revealed the formation of smooth surface spherical nanoparticles. Cellular uptake studies in MIA PaCa-2 (GEM sensitive) and PANC-1 (GEM resistant) pancreatic cell lines confirmed energy dependent clathrin internalization/endocytosis as a primary mechanism of NPs uptake. In-vitro cytotoxicity studies confirmed the hNTs independent transport of GEM in MIA PaCa-2 and PANC-1 cells. Moreover, DNA damage and annexin-V assay revealed significantly higher apoptosis level in case of cells treated with GEM-BSA NPs as compared to free GEM.

Conclusions

GEM-BSA NPs were found to potentiate the therapeutic efficacy by altering physicochemical properties, improving cellular uptake and stability of GEM and thus demonstrated promising therapeutic potential over free drug.

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Abbreviations

6-CFDA:

6-carboxyfluorescein diacetate

8-OHdG:

8-hydroxyguanosine

AFM:

Atomic force microscopy

AnnCy3:

Annexin V-Cy3.18 conjugate

BSA:

Bovine serum albumin

C-6:

Coumain-6

CD:

Circular dichroism

CDA:

Cytidine deaminase

CLSM:

Confocal laser microscope

CNTs:

Human concentrative nucleoside transporters

dFdU:

2′,2′-difluorodeoxyuridine

DMEM:

Dulbecco’s Modified Eagle Medium

DMSO:

Dimethyl sulfoxide

DSC:

Differential scanning calorimetry

EDC:

1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide

EDTA:

Ethylene diamine tetra acetic acid

ENTs:

Human equilibrative nucleoside transporters

FBS:

Fetal bovine serum

FTIR:

Fourier transform infrared

GEM:

Gemcitabine

GEM-BSA NPs:

Gemcitabine-Bovine serum albumin nanoparticles

HBSS:

Hank’s Buffered Salt Solution

hNTs:

human nucleoside transporters NMR: Nuclear magnetic resonance

MALDI-TOF:

matrix assisted laser desorption ionization time of flight

MTT:

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide

NHS:

N-Hydroxysuccinimide

PDI:

Polydispersity index

PXRD:

Powder X-ray diffraction analysis

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

SEM:

Scanning electron microsc opy

SPARC:

Secreted protein acid rich in cysteine

TGA:

Thermogravimetric analysis

TNBS:

2,4,6-trinitrobenzenesulfonic acid

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ACKNOWLEDGMENTS AND DISCLOSURES

The authors are thankful to the Director NIPER, James Graham Brown Cancer Center (University of Louisville, KY, USA) and Strathclyde Institute of Pharmacy & Biomedical Sciences (University of Strathclyde, Glasgow, U.K.) for necessary infrastructure and facilities. Varun Kushwah is also grateful to the Council of Scientific and Industrial Research (CSIR), GOI, New Delhi, United States-India Educational Foundation, New Delhi and Commonwealth commission in the UK for providing research funding and fellowships. He was the 2016–17 Fulbright-Nehru Doctoral and 2015–16 Commonwealth Split-site PhD research fellow at University of Louisville, Louisville, KY and University of Strathclyde, Glasgow, U.K., respectively.

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Authors and Affiliations

  1. Centre for Pharmaceutical Nanotechnology, Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research, SAS Nagar, Punjab, India

    Varun Kushwah, Ashish Kumar Agrawal, Chander Parkash Dora & Sanyog Jain

  2. James Graham Brown Cancer Centre, University of Louisville, Louisville, Kentucky, USA

    Varun Kushwah, Ashish Kumar Agrawal & Ramesh C. Gupta

  3. Strathclyde Institute of Pharmacy & Biomedical Sciences (SIPBS), University of Strathclyde, Cathedral Street, Glasgow, G4 0RE, UK

    Varun Kushwah, David Mallinson & Dimitrios A. Lamprou

  4. Medway School of Pharmacy, University of Kent, Medway Campus, Anson Building, Central Avenue, Chatham Maritime, Chatham, Kent, ME4 4TB, UK

    Dimitrios A. Lamprou

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  1. Varun Kushwah
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Correspondence to Sanyog Jain.

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Kushwah, V., Agrawal, A.K., Dora, C.P. et al. Novel Gemcitabine Conjugated Albumin Nanoparticles: a Potential Strategy to Enhance Drug Efficacy in Pancreatic Cancer Treatment. Pharm Res 34, 2295–2311 (2017). https://doi.org/10.1007/s11095-017-2238-8

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  • DOI: https://doi.org/10.1007/s11095-017-2238-8

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