Encapsulation and systemic delivery of 5-fluorouracil conjugated with

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Encapsulation and systemic delivery of 5-fluorouracil conjugated with silkworm pupa derived protein nanoparticles for experimental lymphoma cancer Naveengandhi Renitta, Berchmans Thiyonila, Veeranarayanan Surya Aathmanathan, Thangaraj Ramya, Natesan Subramanian, Vijay Kumar Prajapati, and M Krishnan Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00404 • Publication Date (Web): 08 Aug 2018 Downloaded from http://pubs.acs.org on August 9, 2018

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Bioconjugate Chemistry

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Encapsulation and systemic delivery of 5-fluorouracil conjugated

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with silkworm pupa derived protein nanoparticles for experimental

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lymphoma cancer

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Naveengandhi Paulin Reneeta1, Berchmans Thiyonila1, Veeranarayanan Surya

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Aathmanathan1,

Thangaraj

Ramya1,

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Ponnusamy

2

Chandrasekar3

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Subramanian , Vijay Kumar Prajapati , Muthukalingan Krishnan *

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1

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2

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Department of Environmental Tiruchirappalli, Tamil Nadu, India.

Natesan

1,2

Biotechnology,

Bharathidasan

University,

Department of Biochemistry, School of Life Sciences, Central University of Rajasthan, Bandarsindri, Kishangarh, Ajmer 305817, Rajasthan India. Department. of Pharmaceutical Technology BIT Campus, Anna University Tiruchirappalli – 620024.

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Word count- 6,297

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Running title- Silkworm pupa derived nanoparticle for delivery of cancer drug

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Corresponding author* Prof. Muthukalingan Krishnan Dean, Academic Sciences Department of Biochemistry Central University of Rajasthan NH-8, Bandarsindri, Ajmer Rajasthan Pin- 305817 India Phone: +91 9443998251 Email: [email protected]

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Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract

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Protein-based drug delivery systems have an edge over conventional drug

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delivery systems due to its biodegradability, non-antigenicity and excellent

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biocompatibility to improve the therapeutic properties of anticancer drug. This study

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describes the increased anticancer efficacy of 5-Fluorouracil (5-FU) conjugated with

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silkworm Bombyx mori pupal bio-waste derived nanoparticles. Here, we have checked

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the toxicity of pupa-protein nanoparticles (PpNps) and its potential as a carrier for

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anticancer drug. PpNps were prepared by desolvation method which resulted in a

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uniform particle size of 162.7 ± 2.9 nm. The 5-FU loaded PpNps was formulated and

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evaluated for its characterization. The drug content of the developed 5 FU conjugated

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nanoparticles was evaluated by HPLC analysis. The entrapment efficiency and

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loading capacity of 5 FU was analyzed by HPLC and showed that 93% and 88.6%

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respectively. The release studies showed the biphasic release of 5FU at pH 7.4 where

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the rapid drug release was achieved for first 30 min, followed by a sustained release

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of 5FU from the developed Nps were achieved for the next 8 h. Mice with developed

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ascites tumor were intraperitoneally treated with 5-FU-PpNps and sacrificed. There

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was a significant increase in total RBCs and hemoglobulin in 5FU-PpNps treated

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mice whereas significant decrease in WBCs indicates the reduced inflammation of

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cancer. Subsequently, 5FU-PpNps decreased the tumor volume and tumor cell

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viability, which proved its cytotoxic property to cancer cells. This study presents a

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novel approach to derive B. mori pupal protein nanoparticles, which can be safely

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used for the cancer drug delivery.

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Keywords- 5-Flurorouracil, nanodrug, anticancer, Bombyx mori, pupal protein

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Introduction

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The smart drug carrier consists of a versatile drug delivery system in which the

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drugs or active materials are dissolved, dispersed, entrapped, encapsulated or attached

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to the carrier1. The drug carrier plays an important role in the target specific drug

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delivery and promoting drug efficacy. The ultimate aim for entrapment of drugs into

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nanoparticles results to enhance the delivery to target cells and moreover reduce in the

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toxicity of the free drug to non-target sites. Conventional methods investigated for

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drug delivery including soluble polymer, liposomes, hydrogel, dendrimers and

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micelles have some limitation, such as wide particle size distributions, poor solubility

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and stability, low drug encapsulation efficiency, fast burst release of drug into the

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extracellular fluid difficulty in site-target specific functionalization and lack of

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tuneable triggering effect of cells for drug release 2,3.

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For these concerns, nowadays materials like chitosan, dextran, alginate, polylactic

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acid, polyethyleneimines starch as well protein which contains collagen are some of

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the natural materials efficiently used as a carriers or gene /drug delivery

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carriers can be processed for both spatial and temporal delivery of bioactive

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substances. Nanoparticles (NPs) are solid colloidal particulate systems ranging in size

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from 1 to 1000 nm and contain nanomolecular particles6,7.

4,5

. These

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Nanoparticles, because of their nano size, allow efficient uptake by various kind

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of cells and also, they can erupt through the endothelial cells in inflammatory sites

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like liver, intestinal tract. By possessing permeability and retention they promote

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quick and target specific drug delivery8,9. The use of these colloidal Nps is effectively

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protecting the drug from a critical environment or their unfavourable bio-

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pharmaceutical mask properties to enhance the stability of the hydrophobic drug and

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absorption to a target tissue. Nps encapsulated with an altered formulation of drug

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designs offer a safe effective alternative to chemotherapy 10,11. So, the researchers are

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precisely involving in creating a new Nps suited for a variety of existing drugs in the

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field of medicine to improve the biodistribution, pharmacokinetics, drug efficacy,

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retention time, the onset of drug-associated adverse side effects and immunogenic

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properties

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unprecedented potential importance due to its unique function over the soluble

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formulation, biodegradable, non-antigenic, intracellular delivery, cell-specific target,

12,13,14

. Protein Nps (PNps) used for the therapeutic purpose have obtained



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low toxicity sustainable release, excellent biocompatibility thermodynamically stable,

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to enhance the drug solubility and permeability 15,16. Especially PNps are made up of

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multiple proteins-based building blocks linked either by protein-protein or protein-

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ligand interaction exhibit various functional groups

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altered by covalent attachment of ligands or drug for better therapeutic efficiency 18.

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The PNps allow the electrostatic adsorption of positively or negatively charged

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molecules without the demands of another compound. So, they can be easily prepared

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either desolvation or by coacervation processes 19.

17

. The surface of PNps can be

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Proteins are dynamic macromolecules that have the capacity to possess a complex

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unique character which makes it a potential therapeutic agent. However, it is easy to

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understand the architecture, cellular and molecular physiology, physiochemical

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properties, proteolysis, denaturation condition and molecular recognition 20,21. Protein

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encapsulated nanoparticles with drug possess the non-antigenic specific capacity and

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exhibit various functional groups that can easily penetrate to a biological response

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host cell, alterations of specific enzyme activities or apoptosis regulation, affinity

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targeting 22. This property makes them as the suitable candidates for drug delivery.

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Silkworm pupa (Sp) are natural enormous by products of the silk reeling industry.

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Sp is the protein bio-macromolecules which contain high-quality of protein and all

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essential amino acids namely methionine, valine, and phenylalanine. Apart from high

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protein content, it is also a sole source of omega-3 fatty acids, particularly Alpha -

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Linolenic Acid (ALA), DHA, and EPA23,21. It has been extensively used in the

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biomedical fields as a bio-material in of tissue engineering, tissue scaffolds,

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regulation of blood lipids and glucose level, and wound dressings. Especially its

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biocompatibility, biodegradability, haemostatic, bioreactor, controlled drug release,

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chemical modification potential and cross-linking efficiency make the SP- Nps as a

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promising drug delivery system24.

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In this study we aim to formulate, develop and optimize a novel drug carrier

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system for 5-Fluorouracil using silkworm pupae derived nanoparticles. Though 5-FU

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has been investigated in various aspects of Nps encapsulation but there is no reported

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literature on protein pupae Nps (PpNps) which can directly use as a delivery vehicle

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for 5-FU. In this method, we report, the formation of PpNps using desolvation

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method25. The formulation aspect of this study to elucidate the effect of as a carrier


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for the drug, stabilizer concentration and emulsifier effects of overall drug loaded and

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release characteristic. The cytotoxic and anti-cancer efficacy of 5-FU-PpNps was

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investigated in both in-vitro and in-vivo.

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Result and discussion

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Characterization of PpNps

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The interaction of protein Nps may induce protein conformational changes.

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The changes in proteins may expose unknown epitomes and subsequently activate

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undesired signaling pathways

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conformational structure when Nps interact with proteins. FT-IR was used to identify

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variations in chemical functional groups present in the protein Nps. It provided

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information about, its possible secondary metabolites responsible as capping and

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reducing agent for protein Nps are shown in Figure 1. The FTIR peak of protein Nps

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prepared by using desolvation method were observed at 2265cm-1 indicate the

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possible interaction of aromatic residue (tryptophan) C-O-C group. The peaks at 2958

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cm–1, at 2932 cm-1 and at 2873 cm-1 were assigned, activated to be functional groups.

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The variations in the absorbance at 1700cm-1, 1600cm-1, 1630cm-1, 1500 cm-1,

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1512cm-1 are important amide bands

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The pupa spectra were observed at 2957 cm-1 are belongs to CH stretch of alkanes

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group, an asymmetric stretch of C−H found at around 1238 cm−1and the peak at 1317

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cm–1 belongs to the C−H vibration in peptide bonds. The peak around 1938 cm-1-

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1025 cm -1 due to symmetric vibrations of CH=CH that are present in carboxylic acid

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dimer OH group. Distinct peaks at 1295 cm-1 and 1266 cm-1 are due to amide II

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vibration (1800-1700 cm-1) of protein.

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. Therefore, it is crucial to monitor protein

27

which confirms the present of protein Nps.

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The stability and distribution particle size of protein Nps were measured using

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a Nano-zeta size (Malvern UK). The average particle size was approximately 162.2 ±

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2.9 nm, and its PDI value was 0.250 ± 0.042 respectively (Figure 1b and c and

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Table 1). The zeta potential value was 34.7 ± 1.7 mV, which shows the particles are

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positively charged, dispersed in the solution with good stability and showed the

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electrostatic repulsion between them. Further, the morphology of the protein Nps was

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shown in Figure 1d. The core size and morphology of Nps were revealed with TEM

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analysis. The Nps nearly spherical in nature and the particle size ranges from 30-60

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nm. In order to study the crystalline nature of protein Nps, the Selected Area Electron



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Diffraction (SAED) analyses were carried out at a different region of the particles.

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The ring-like diffraction pattern (SAED) image shows that particles are purely

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crystalline in nature

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Hemolysis assay:

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To assess the impact of PpNps on erythrocyte, hemocompatibility test was

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performed by spectrophotometric measurement of hemoglobin release after exposure

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to various concentrations of PpNps. Triton X-100 used as a positive control exhibited

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100% complete lysis in the Red Blood Cells (RBC’s) whereas PBS used as the

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negative control and PpNps show 10 % toxicity to the RBC (Figure 2). No adverse

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reactions were observed between the serum proteins and the surface of the

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nanocarrier. Our results revealed that the tested concentrations exhibited neither

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hemolytic activity nor thrombus formation which make them suitable for circulation

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in the blood and it’s provoke to be promising clinically formulation. The results were

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consistent with results obtained by 28

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Invitro proliferation effect of PpNps on peripheral lymphocytes

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Cytotoxicity, the most sensitive test for estimation of the cells viability was

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measure using MTT assay. The lymphocyte proliferative responses were investigated

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to assess the effects of PpNps on the basic immunological functions of human

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lymphocytes. The cells viability was measured using Peripheral blood monocytes

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(PBMCs), after exposure to various concentrations of PpNps. The cells that are viable

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after 24 h of exposure to PpNps were capable of metabolizing a dye efficiently and

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the purple colored precipitate was analyzed. It was clearly demonstrated that PpNps

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was non-toxic to PMBC at the selected concentration range. The lack of any

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noticeable toxicity of PpNps provides new opportunities for safe application and used

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a carrier for cancer drugs were shown in supplementary Figure 1.

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Encapsulation efficacy and release entrapment

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Encapsulation efficiency (E) of the PpNps was measured by calculating the ratio

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of released proteins in the aqueous solution as explained in the method section. About

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88% of encapsulation was achieved by using desolvation method used for protein-Nps

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fabrication. Although, there was an increase in total protein encapsulation which

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resulted decreased in the encapsulation efficacy (Table 2). A cumulative release was

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measured over the period of 6 days in phosphate saline solution. The burst release of


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protein (biphasic) was observed over the period of first 24–48 h and minimal

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sustained release was observed in the following subsequent days was shown in

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supplementary Figure 2). The increase total protein content associated with the faster

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rate of release during the sustained-release phase could serve as a versatile drug

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delivery carrier system.

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Conservation of the entrapped and integrity of PpNps

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The possible change in pupa associated protein Nps during pre and post

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encapsulation were subjected to electrophoretic analysis on 12% SDS-PAGE. The

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relative predominant CBB stained protein bands were observed between 15 to 250

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kDa in pupa extract. However, no significant different between the pupa extract and

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PpNps was noted but reduction in intensity of major protein bands resolved at 180

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kDa (vitellogenin) and 74–80 kDa (storage protein; SP1, 2 and 3) when compared

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with the pupa extract were observed in Figure 3. PpNps interact more strongly with

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vitellogenin and storage protein which are responsible for lipid transport and energy

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metabolism possess, a dysopsonic effect. Further, they initiate receptor-mediated

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endocytosis pathway for intracellular transport of these nanocarrier in vivo with

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hydrophobic interaction to form protein corona. These proteins were previously

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isolated and characterized by our laboratory using mass spectrometry analysis. 29,30.

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Drug loading, entrapment and in-vitro release kinetic

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The entrapment, drug loading efficiency and release kinetics of the drug

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conjugated PpNps were analyzed using a HPLC. The half-life of 5-FU invitro was

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found to be 5 to 10 min. Thus, many studies have attempted to develop 5-FU

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conjucated with Nanocarrier for a prolonged lifetime

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concentration was found by correlating the absorbance OD of the supernatant after

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conjugated with standard absorbance OD. The drug entrapment and loading efficiency

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of 5FU were found to be 93% and 88% respectively. For determining the amount of

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drug released from the nanoparticles was studied using PBS, the absorbance values

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were recorded using a HPLC at 254 nm for the samples taken at regular time

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intervals. The released amount of drug was estimated using derived calibration curve,

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the absorbance values, and the entrapment efficiencies. There was a steady release of

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drug in the early hours and a total release of about 90% was observed (Figure 4).The

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time depended cytotoxic effect of PpNps allowed these drug to evenly distributed in

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. The free or unbound drug


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the body, thereby increasing the half-life period and decreasing its toxic side effect

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on normal tissue.

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Covalent immobilization and release

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The PpNps prepared by using Two-step desolvation method, before and after

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conjugated with 5FU were observed using FTIR analysis. The peak at 3389 cm-1

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assigned to have possible stretching vibration of OH (amide A), peak at 2971 cm-1

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that assigned to be C–H (alkanes compounds) stretching vibration, at 1295 cm-1 that

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assigned to have II amide group (Ar N) (Figure 5a). The FTIR peaks of PpNps after

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conjugated 5FU were observed at 1747 cm-1 that assigned to be amide I (mainly C=O

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stretching vibrations), 1676 cm-1 that assigned to be amide II (C=O stretch), at 1157

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cm-1 that assigned to have amines (C-N stretch).The two predominated peaks were

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observed at 1374 cm-1, 1430 cm-1 and were found to be overlapping with each other,

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which confirmed the functional group interaction with C=C stretch alkenes compound

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and –C-O stretch carboxylic acid of PpNps and drug. Further, the covalent linkage of

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5FU to PpNps was demonstrated by SDS-polyacrylamide gel electrophoresis (SDS-

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PAGE). The SDS gel reveals a mobility shift of 5FU bound to PpNps when compare

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to that of free 5FU. The PpNps -5FU conjugate migrates slightly slower than PpNps

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due increased in molecular weight (Figure 5b).

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Non-covalent interaction in drug loading and release

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The cellular uptake of 5FU conjugated PpNps starts early in 1 h to 4 h of

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treatment. The in vitro release of drug from PpNps was investigated through the acid-

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dependent hydrolysis linker simulated in the range of pH reflect early endosomes (pH

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6.0), late endosomes (pH 5.0) and lysosomes (pH 4.5) The sustainable release of 5FU

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in an acidic environment, similar to that of physiological damage or target tissue in

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the human body at 37°C. 65% of the 5FU conjugated to the interior of PpNps was

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released after 2 hours at pH 4.5 but only 25% and 33% was release after 2 hours at pH

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5.0 and 6.0 which indicates the sustainable release in the membrane of the cells and

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function accordingly. The 5FU conjugate onto PpNps by acidic pH linkage severed

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upon acidification in the endosomes and lysosomes followed by uptake in the

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endocytic pathway. 5FU release from the drug delivery carrier enters into the nucleus

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in a time-dependent manner (Figure 6). At low pH condition, the drug release from

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the nanoparticle conjugated system in the endosomes coupled to enhanced


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permeability and retention effect (EPR) in the cancerous tissues to ensure selective

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toxicity only on those cells. However, by using of natural biopolymer and acid

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triggered the release of 5FU has been shown effective both in vivo and in vitro 32. This

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study could provide better knowledge about the release and effect of 5FU conjugated

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PpNps under the extracellular acidic condition in an animal model for tumor therapy

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under the biologically under lysosomal condition

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Environmentally -triggered structural changes of PpNps:

. Our data show the potential of 5FU released from protein conjugated nanoparticles

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The present study mainly focused on nano-biointeraction on environmental

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condition by using various parameters like temperature, alkaline pH and ionic salt

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condition which may directly effect on cellular response. Thermos-sensitive protein-

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drug complex was engineered using the desolvation method are mainly influence on

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particle size and encapsulation efficiency. When the temperature was decrease to

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25°C instability of particles with minimum size of 75 nm and decreased in loading

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efficiency was obtained. On increased to 37°C moderate particle size of 167.8 nm

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with good stability was obtained and influence a successful loaded with drug Nps

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complex and maintained at normal body temperature as evidence by drug retention at

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37 °C for long period of time. The largest size particles of 364.56 were obtained at

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42°C which shows moderate dependent poor stability with modulation of mild

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incorporation of PpNps with drug were obtained. The changes in alkaline pH play a

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major role in triggering the structure of Nps. With increased in pH, the mean diameter

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size of Nps decreased and significantly increase the percentage of yield percentage.

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On comparing the alkaline pH (8.5,9.5,10.5), the optimal pH was found to be 9.5 with

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the average mean diameter of 168.2 nm and with the encapsulation efficiency of 88%.

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The ionic salt concentration plays a key role in optimization process and yield of

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controlled size particles. However, with a decreased in the ionic concentration of

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NaCl (3.5 mM,4.5 mM,5.5 mM) may not greatly influence on mean diameter size and

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yield percentage. The size of the particles may slightly fluctuate therefore the particles

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size are in the range of 165 to 250 nm were shown in Table 3

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Antiproliferative activity of 5FU and PpNps conjugated 5FU on colon cancer cell

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line

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The mammalian HT-29 cells line was used to elucidate the anti-proliferative

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activity of different concentration of 5FU-PpNps and 5FU. The result shows that



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antiproliferative effect of 5FU-PpNps on HT-29 shown in supplementary Figure 3

289

was significantly higher than that of free 5FU. It also been observed that 5FU-PpNps

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conjugate inhibited the cell proliferation even in the lower concentration, suggested

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that they may be used as the suitable drug carrier to reduced systemic toxicity and

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adverse effect of drug.

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Ex vivo analysis

294

As determined by Ex vivo analysis, 40 µg /kg /Bw of standard 5FU was found to

295

have the inhibitory effect towards 50% of tumor cell (IC50 value) by using trypan blue

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dye exclusion assay for live and dead cells. The IC50 value was found to be 35 µg /kg

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/Bw for PpNps. For 5FU conjugated PpNps IC50 value was found to be 30 µg /kg

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/Bw.

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Tumor mass inhibition Study

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The DAL cancer cells (1x 106 per mouse) were injected into mice to study tumor

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growth inhibition by invivo. Tumor volumes were measured using calipers scale on an

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interval of 4 days until study completion. Initially, no much significant difference in

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tumor size volume was observed between control and treated group. After the

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incubation period, increased tumor growth size was observed. Even, though PNps was

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initially efficient in suppressing the tumor growth than 5FU, it did not show much

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effective for a long period of time due to non-bioavailability of molecules in the

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bloodstream. The 5FU conjugated PpNps showed a significant reduction in tumor

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growth when compared to the normal standard drug. The reduction in tumor size by

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5FU conjugated PpNps nanoparticles was shown in Figure 7. Interesting fact that o

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5FU-PNps treated mice groups did not show any considerable variation in weight loss

311

and the animals are active during the experimental studies

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Effect on life span increases in tumor induced mice

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The effect of Mean Survival Time (MST) was calculated for all five groups. The

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increase life span was calculated on basis of ascitic suspension volume elevated in

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tumor induced group when compare to test treated group. The increased MST was

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observed from 22.45 ± 3.20 to 38.43 ± 4.35 in the tumor induced mice when treated

317

with 5FU-PpNps group. The life span was effectively increased in test group as

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compared to 5FU group. Similarly, significant increase in MST was observed in 5FU

319

(standard drug), PpNps and 5FU-PpNps treated groups when compare to DLA disease

320

group mice (Figure 8)


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The difference in the body weight gain in mice

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Body weight measurement is a direct indicator of ascites tumor growth and

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development. Due to the accumulation of ascites suspension in the peritoneal cavity,

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the significant increase in body weight with the sluggish movement was observed in

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DAL induced mice when compared to the normal animal. The changes in the body

326

weight were observed on every 4 days time interval from the period of induced and

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until the results obtained are shown in Figure 9. The standard drug and 5FU-PpNps

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treated mice s from 1st to 14th day of induction showed a decrease in body weight

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when compared to tumor-induced. These finding suggest that, they may prevent the

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gastrointestinal hemorrhage side effect associated with drug that are responsible for

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reduced in body weight.

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Effect on ascitic tumor growth and cell viability

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Cell viability assay is an important method for toxicology analysis which

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response to cellular level of toxic materials. DAL induced mice showed a tremendous

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increase in ascitic suspension volume, which is required nutrition for growth and

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development of tumor cells. When treated with drug conjugated PpNps showed a

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maximum significant decrease in the ascitic volume suspension, cell viability and

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subsequently arresting tumor development when compared to standard. (Figure 10 a,

339

b ). On 14th day, maximum inhibition was achieved in test group animals when

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compared to induced groups

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Effect of 5Fu-PpNps on angiogenesis

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Neovascularization is a key factor in the growth and development of tumor

343

cells. It generates various pro-angiogenic factors to promote the migration,

344

proliferation and tube formation on endothelial cells

345

observed in the DAL induced group shown in Figure 11. Therefore, after the

346

treatment with 5Fu-PpNps, blood vessel abowman’s capsule. While the hepatic and

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renal structure of the mice administered with 5FU showed a slight enlarge, moderate

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sinusoidal dilatation and slight remarkable different in cells were appear in glomeruli

349

and bowman’s capsule of kidney. Whereas the group administered with 5FU

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nanoconjucated PpNps revealed markable improvement in the histological structure

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similar to that of control

352

Evalution of Histomorphometrical changes in the tissue

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. Increased angiogenesis was



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Histopathological examination of liver and kidney sections of experimental and

354

control group were examinated using H&E staining. The control group exhibited

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normal arragement on hepatocyte vacuolar and renal corpuscles with regular

356

sinusodial dilatation and glomerulus bowman’s space were shown in supplementary

357

Figure 4 and 5.Whereas enormous pathological alterations were observed in DAL

358

induced group, an enlarged severe necrosis on hepatocytes, severe infiltration of

359

inflammatory cells around the portal vein, moderate sinusoidal dilatation and

360

distortions of renal tubules, congested vein were appear in the glomerular space and

361

bowman’s capsule. While the hepatic and renal structure of the mice administered

362

with 5FU showed a slight enlarge, moderate sinusoidal dilatation and slight

363

remarkable different in cells were appear in glomeruli and bowman’s capsule of

364

kidney. Whereas the group administered with 5FU nanoconjucated PpNps revealed

365

markable improvement in the histological structure similar to that of control.

366

In vivo biodistribution experiments with 5FU conjugated protein nanoparticles

367

In vivo anti-tumor efficiency was evaluated in the DAL cells induced mice

368

model. The concentration of 5FU was measured in plasma, blood, liver, kidney,

369

spleen, heart, and muscle (Figure 12). Although 5FU are the most effective

370

anticancer drug but it associated with various side effect, such as nephrotoxicity,

371

hepatic toxicity, nausea and emesis

372

PpNps reduced the toxic side effect on normal tissue and act as target specific drug

373

for tumor tissue to achieve high anticancer efficacy by receptor mediated endocytic

374

pathway. Smaller particle size was used for the targeting the tumor cell owing to

375

‘‘enhanced permeability and retention’’ (EPR) effects which resulted in the efficient

376

accumulation in the tumor. Also, the higher distribution percent in the tumor of 5FU-

377

PpNps might be due to the prolonged blood circulation time.

35

. So sustainable release of 5FU conjugated

378 379

Hematological tests

380

In cancer chemotherapy, anemias due to iron deficiency or hemolytic

381

conditions are the major problem associated with cancer progression. So, it was

382

important to study hematological parameters such as Hb, RBC, WBC, HCT, MCV,

383

MCH, MCHC, PLT, neutrophils and lymphocytes in control and experimental mice

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shown in Figure 13 & 14. The animals were sacrificed after the 14th day of treatment

385

and blood was collected from each group of experimental animals, anti-coagulated


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386

used for the estimation of packed cell volumes. Statistically significant changes in

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important hematological parameters were investigated. There was a significant

388

decrease (p

Encapsulation and systemic delivery of 5-fluorouracil conjugated with

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