Preparation And Anti-pancreatic Cancer Efficacy In Vitro Of Gemcitabine-loaded Nanovesicles

Background and objectiveThe malignant degree of pancreatic cancer is very high, and the prognosis is very poor. The incidence rate has a rising tendency. The symptoms of earlier period are not typical. Majority of them have entered the late phase when apparent symptoms occur, and lose the chance of radical cure. The position of pancreatic cancer is deep, and there are stomach, intestines, and spinal cord around it, moreover, its sensitivity to radio- therapy is bad, and its tolerant dosage is low, so the effect of radiotherapy is not ideal. Chemotherapy is the main therapeutic measure for pancreatic cancer of late phase, but majority of the patients with pancreatic cancers belong to the middle or late period when the final diagnoses is established. Gemcitabine has shown significant clinical activity in pancreatic cancer, with significant improvements both in survival and clinical benefit, leading to Food and Drug Administration (FDA) approval of Gemcitabine in advanced pancreatic cancer. Because Gemcitabine disperse all around the body after administration, they kill not only the cancer cells but also the normal cells, which lead to a lot of side effects such as bone marrow suppression and gastrointestinal toxicity,thus limiting the clinical application of Gemcitabine.In the recent years, with the rapid development of nanotechnology, the nanoparticle drug delivery systems received increasing attention in medical field especially in diagnosis and treatment of carcinoma, for its means to obtain a higher therapeutic effect, a lower toxicity and a protection from in vivo metabolization of incorporated drugs.Nanometer is a type of measurement units, a nanometer equals a millionth millimeter. Nanoparticle drug delivery systems are colloidal particles of approximately 10 nanometers to 1000 nanometers in size, in which the drug is adsorbed, dissolved, or dispersed throughout the matrix, and the drug can release by diffusion,penetration and polymer erosion. Nanoparticle drug delivery systems use in the diagosis and treatment of malignant tumor are mainly composed of nanosphere and nanovesicle. As a new drug delivery carrier, nanoparticle drug delivery systems may offer many advantages including the possibility of targeting to specific sites, controlled release of therapeutic agents as well as protection of encapsulated drug.The materials of carriers preparing nanoparticls are all high molecular compound, largely are the synthetic biodegradable polymer system and natural macromolecule. As far as concern, polylactic acid(PLA) have been widely used as carrier materials in nanoparticle drug delivery systems due to their excellent biocompatibility. Their end products of metabolism in human body are hydrate and carbon dioxide. The techniques to prepare drug-loaded PLA nanoparticls mostly include: solvent evaporation, spontaneous emulsification solvent duffusion, salting out/emulsification-diffusion. Among them, double emulsion technique was widely used to prepare nanoparticles loading hydrophilic drugs as follow: first emulsion (w/o) was formed between an organic solution of the polymer(polymer dissolved in a water-immiscible organic solvent) and an aqueous solution(drug dissolved in the pure water ) under a high energy mixing step using a high energy source such as ultrasounds, homogenizers,high pressure dispersers, colloid mills or microfluidizers, and was added dropwise into an external aqueous phase consisting of emulsifier such as PVA under under mixing step to obtain the double emulsion (w/o/w), afterward, the residual organic solvent was completely removed by vacuum distillation with arotary evaporator. Nanovesicles obtained as a suspension were purified by filtration through a syringe filter, and then subjected to lyophilized to yield the solid nanovesicle samples.However, nanoparticles base on PLA accumulate blood proteins on their surfaces as they circulate through the body for the unsatisfied hydrophilicity of PLA,due to PLA containing more hydrophobic ester group reduces the hydrophilicity. This nonspecific absorption of proteins attracts attention from immune system cells, with the result that nanoparticles are often removed from circulation and targets to MPS organs such as liver, spleen, lung, lymphatic. but for non-MPS organs (such as the pancreas), this passive targeting are unfavorable. One way to solve this problem is to performan surface modification, there are two surface modification methods for PLA nanoparticl, one is surface coating technology using hydrophilic polymer or surfactant coating, another is to develop block copolymer of PLA. Polyethylene glycol(PEG) was widely used to carry out the surface modification by synthesizing the PEG/PLA block copolymer due to its excellent hydrophilicity and biodegradability. The PEG chains immobilized on the surface forming a hydrophilic palisade, can create repulsion between each other, this repulsion can stop the nanovesicles from agglomerating, thus increasing the dispersion stability in aqueous media, furthermore, the PEG are able to repel proteins from adhering to the surface and thus avoid the nanovesicles recognition by macrophage cells, whitch lead to prolong the circulation time and to facilitate the nanoparticle uptake to specific cancer cells for cancer therapy.In our research,the nanovesicles as the carrier was prepared from the amphiphilic block copolymer of poly(ethylene glycol)-block -poly(D,L-lactide) by diemulsion technique, and Gemcitabine was used as the model drug. The prepared Gemcitabine-loaded possess long circulation characters by using PEG to perform surface modification.MethodsThe nanovesicles was prepared from the amphiphilic block copolymer of poly(ethylene glycol)-block-poly(D,L-lactide) by diemulsion technique, and Gemcitabine was used as the model drug. The morphology of vesicles was determined by scanning electron microscope (SEM) and transmission electron microscope(TEM), and its drug loading, encapsulation ratio were detected by ultraviolet Spectrophotometer. In order to gain the best parameters for preparing Gemcitabine-loaded nanovesicles, the influences of different factors on average drug loading and incorporation efficiency of Gemcitabine-loaded nanovesicles were evaluated and the optimal preparation parameters were selected by orthogonal-designing method using L9(34) table ; In vitro release profiles of Gemcitabine from nanovesicles were obtained by a dissolution test in phosphate buffer solution (PBS, release medium, 37℃,pH 7.4 and 5.0);Their cytotoxicity on human pancreatic cancer cell line SW1990 was tested by MTT assay.ResultsThe prepared nanovesicles was sphere in shape with hollow structure, with the mean size of 200.6 nm, DL of 4.14% and ER of 20.54%. These nanoparticles show a stable release profile without a manifest burst effect, the release behaviour of gemcitabine from nanovesicles exhibited a biphasic pattern characterized by a fast initial release during the first 72h, followed by a slower and continuous release. The cumulated amount of Gemcitabine released from nanovesicles was less than 30% at pH 7.4 and 92.7% at pH 5.0 after 18 days. The MTT assay demonstrated that gemcitabine-loaded nanovesicles exhibited time and dose-dependent cytotoxicity on human pancreatic cancer cell line SW1990.Conclusions1.The nanovesicles prepared from PEG-PDLLA can be served as one of carriers for Gemcitabine2.The prepared Gemcitabine-loaded nanovesicles possessed good performance of drug controlled-release.3.The prepared Gemcitabine-loaded nanovesicles show excellent cytotoxicity on human pancreatic cancer cell line SW1990.