| Background and objective:Ultrasound targeted microbubble destruction (UTMD) technology is a potential method for drug delivery because microbubbles (MBs) can be disrupted by a localized ultrasound to release therapeutic agents at the targeted site. Under the influence of ultrasound, microbubbles can implode thereby leading to the alternate growing and shrinking of microbubbles (inertial cavitation). Meanwhile, fluid streams and microjets can transiently perforate cell membranes and, therefore, enhance the intracellular uptake of drugs, which is called sonoporation. Although the use of MBs by ultrasound exposure is proved to be highly beneficial in intracellular delivery, the thin MBs shell and gas core each have limited drug-carrying capacity, thus can not yield an efficient delivery vehicle.Paclitaxel, one of the most widely used anticancer drugs for the treatment of various human malignancies, can promote the aggregation and assembly of microtubules and interfere with cellular mitosis, thereby inhibiting tumor cell proliferation. Due to its poor solubility in water, paclitaxel is currently formulated in a1:1mixture of Cremophor EL and ethanol. However, Cremophr EL has been documented for various serious side effects, including myelosuppression, nephrotoxicity, neurotoxicity, cardiotoxicity and acute hypersensitivity reactions. Biomacromolecules have good biocompatibility and biodegradability, in particularly, enhanced permeation and retention (EPR) effect is now considered as a major mechanism for their unique biodistribution profile in the tumor tissue. The distinctive properties have significant implications in the development of biomacromolecules-based nanoparticulate delivery system, which can improve drug solubility, increase loading capacity as well as permit drug controlled release, etc. In this study, we used a biocompatible, biodegradable, and water-soluble natural polysaccharide heparin as a carrier to entrap hydrophobic paclitaxel thereby forming heparin-based nanoparticles, which can reduce its side effect and enhance its solubility, etc.Although benefited from the EPR effect, the unspecific accumulation of nanoparticles has also been observed due to their classical size effect. Combination with the properties of MBs and nanoparticles, we designed strategy for the attachment nanoparticles to MBs by avidin-biotin bridge, which is expected to open many exciting opportunities for the targeted delivery of therapeutic agents to cancer as well as the use of US as a noninvasive strategy to enhance drug uptake and monitor targeting efficiency to improve the therapeutic outcome.Endothelial barrier and the cell membrane is the main barrier for gene and drug delivery. Meanwhile, an inevitable problem in cancer treatment is that shortage of lymphatic drainage in tumor tissue causes the interstitial pressure increase thereby arresting drug penetration within cancer cells. Sonoporation under UTMD technology can not only enhance endothelial gap and membrane permeability, but also improve therapy efficiency. Despite of unique properties of UTMD, drugs enter into cell randomly, so the main problem need to be solved is how to make drugs positively transfer into the endometrial barrier and concentrate into cells thereby increasing effective drug concentration.Cell Penetrating Peptides (CPPs) consist of10to30amino acids possessing the ability to transfer various cargos such as proteins, antibodies, nucleic acids, liposomes, imaging agents and cytotoxic drugs, etc, across biofilm barriers (including the blood-brain barrier) into cells. The application of CPPs has provided effective method for diagnosis and treatment of disease. However, one of main obstacles that still remain unresolved is that CPP does not possess targeting selectivity in cell entry, significantly hindering its potential applications in vivo. Folic acid (FA) can selectively bind to folate receptor (FR), a cell surface receptor, which is significantly upregulated in breast, ovary, and endometrium cancers as compared to that in normal tissues. Hence, the combination of target ligand-mediated selectivity and CPP-mediated effective transmembrane function may be a good strategy for production of new drug delivery system for further application. Herein, we first fabricate dual-functionalized NPs composed of heparin-based polymer conjugated with folate and Tat peptide to differentially deliver paclitaxel. Also, Heparin-Folate-Tat is biotinylated by using biotinamide convenient for the combination with the ultrasound-mediated delivery. Subsequently, dual-functionalized nanoparticles are incorporated with microbubbles by avidin-biotin binding of nanoparticles to the lipid shell of nicrobubbles. Paclitaxel delivery by such construction can be attractive, as it would take profit of biological ligand effects from nanoparticles and sonoporation effect from microbubbles. Only in response to acoustic pulse, nanoparticles are released from complexs and nanoparticles extravasation can occur at the targeted site by UTMD. The FR-mediated endocytosis, Tat-mediated transmembrane function and ultrasound-mediated effect together contributed to the enhancement drug uptake by combining functionalities simultaneously and specifically to tumor. Materials and methods:1. Synthesis and characterization of DiI-labeled microbubblesThe ultrasound contrast MBs were prepared according to the method as follows. Briefly, some amount of DSPC, DPPE-Biotinlyated PEG4000and DiI dye in20mL of water were stirred at70℃for30min and then were cooled down at room temperature. The mixed lipid suspension was sonicated by a shear cutter and then perfluorobutane gas (C3F8) was slowly injected into solution under a certain shear rate for2min. The DiI-labeled lipid microbubbles were sealed into schering bottles. The size and concentration of MBs were determined by a Coulter counter. Morphologic characteristics of DiI-MBs were determined under a fluorescent microscope and confocal microscope.2. Preparation and characterization of Biotinlyated Heparin-Folate-Tat-Paclitaxel nanoparticles (Biotinlyated H-F-Tat-P NPs).2.1. Material preparation(1) Synthesis and characterization of Tat peptideTAT peptide of the sequence Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg was synthesized by ChinaPeptides CO., LTD. The purification of peptide was conducted by column chromatography and purity of peptide was determined by high performance liquid chromatography (HPLC) methods.(2) Synthesis of Succinylated-heparin.Succinylated-heaprin was synthesized as follows. Briefly, certain amount of heparin sodium salt dissolved in water was percolated through a732column (H+form). The pH of the solution was adjusted to6.0by addition of tetrabutyl ammonium hydroxide. Excess ammonium was eliminated by evaporating. The concentrated solution was diluted with water and lyophilized to yield tertrabutyl ammonium salt. The tertrabutylammonium salt was dissolved in dry DMF and was then cooled down to0℃. Certain amount of succinic anhydride, triethylamine, and4’,4-dimethylaminopydine (DMAP) were successively added and the reaction was allowed to proceed at room temperature for24h. Excessive solvent was evaporated and water was added to the flask followed by passing through a732column (H+). Then the product was dialyzed and the effluent was neutralized with and lyophilized as a white powder.(3) Preparation of biotin-NH2Certain amount of D-Biotin and N-hydroxy succinimide (NHS) in DMF was mixed and stirred under70℃until reactants completely dissolved and cooled down to room temperature. Subsequently, catalytic amounts of N, N’-dicyclohexycarbodiimide (DCC) was added to the solution and the reaction was allowed to proceed at room temperature for24h. The reactant was filtered and the supernatant was collected. Certain molar ratio of triethylamine and ethylenediamine were added into supernatant and reacted for3-4h. The reactant was added to excessive ether and the precipitate was collected and centrifuged. Biotin-NH2was obtained as a white yellow powder. (4) Preparation of folate-NH2Certain molar ratio of folate, DCC and NHS in DMSO was reacted for5-6h at50℃. The reactant was filtered and the supernatant was collected. Subsequently, certain molar ratio of triethylamine and ethylenediamine were added into supernatant and reacted for24h. After reaction, excessive acetonitrile was added to the supernatant and precipitation was collected and dissolved in deionized water. After pH of the solution was adjusted to6, the solution was filtered and washed by anhydrous ethanol and diethyl ether. Folate-NH2was obtained as yellow powder.2.2. Preparation of Biotinlyated Heparin-Folate-Tat-Paclitaxel NPs (Biotinlyated H-F-Tat-P NPs).Succinylated-heparin, folate-NH2, Tat peptide, biotin-NH2, Oregon green, EDC and NHS were mixed, and the reaction was allowed to proceed at room temperature. After overnight stirring, paclitaxel was added to the reactant for30min, and then cold deionized water was slowly added to the solution. Reacted residues were removed by a dialysis membrane for48h. The excess water was evaporated and resulting aqueous solution was filtered through a450nm membrane. The nanoparticle suspension was finally lyophilized as a light yellow powder. The nanoparticle structure was measured by1H NMR and the morphology of nanoparticles was determined by transmission electron microscope (TEM). The particle size and zeta potential were measured by dynamic light scattering instrument. The content of drug in nanoparticles was determined by UV method.3. Preparation of Biotinlyated Heparin-Folate-Tat-Paclitaxel NPs loaded with Microbubble (NPs-loaded MBs).NPs-loaded MBs was prepared as follows. The biotinylated microbubbles were incubated with avidin at room temperature with gentle shaking for10minutes. After purification by washing, biotinylated H-F-Tat-P NPs was mixed with avidinylated microbubbles and incubated in the ice bath for30min with gentle shaking. MBs carrying NPs were washed and purified to remove free NPs. Oregon green labled H-F-Tat-P NPs loaded with Dil labled microbubbles was visualized using a confocal laser scanning microscope to investigate the binding efficiency and construction of NPs-loaded MBs. The amount of NPs bound to MBs was estimated by removing the unbound NPs from MBs via centrifugation.4. Cellular uptake experiments4.1. The optimization of ultrasonic irradiation conditionsMDA-MB-231cells and A549cells were employed as FR overexpressing and deficiency cancer cells treated with NPs alone and NPs-loaded-MBs with and without US (Oregon green labeled NPs). The cells were incubated with NPs-loaded MBs and exposured to ultrasound with different radiation intensity and radiation time. After4h incubation, the cells were washed three times with cold PBS to eliminate trace product and fixed by4%(w/v)para-formaldehyde solution. The fluorescent images were viewed by confocal microscope.4.2. Quantitative analysis of cellular Uptake of NPs and NPs-loaded MBs with and without US by flow cytometry:The cells were incubated with NPs (Oregon green labeled H-F-P NPs, H-Tat-P NPs, H-F-Tat-P NPs) and NPs-loaded MBs with or without ultrasound condition. After4h incubation, the cells were washed three times with cold PBS to eliminate trace product and detached with0.02%EDTA-PBS and then suspended in PBS containing0.1%BSA. The suspended cells were directly introduced to a FACSort flow cytometer equipped with a488nm argon ion laser.4.3. Qualitative analysis of cellular Uptake of H-F-Tat-P NPsOregon green labeled H-F-Tat-P NPs and NPs-loaded MBs were incubated with two cell lines under ultrasound condition. After4h incubation, the cells were washed three times with cold PBS to eliminate trace product and fixed by4%(w/v) para-formaldehyde solution. The fluorescent images were viewed by confocal microscope.5. Cell viability assay5.1. Blank MBs, paclitaxel, NPs and NPs-loaded MBs with and without were incubated with two cells, at final drug concentration of9,18,36and54μg/mL for24h, respectively. MTT assay was used to measure the relative cell viability and determine IC50. 5.2. Statistical analysis.Statistical analysis was performed using the SPSS13.0software package. All data were performed in triplicate and presented as a mean value with its standard deviation indicated (mean±SD.) Difference of cell viability in different drug delivery system groups were analyzed with one-way ANOVA. If heterogeneity of variance was observed, the Welch-corrected t test was used to calculate P values evaluating the significance of differences in group means. P<0.05was considered statistically significant.Results:1. The mean diameter of MBs was2.20±0.93μm and the concentration was (11.31±1.0)×108/mL as determined by Coulter Multisizer. The microbubbles had a good shape and uniform distribution without aggregation under the light microscope. The microscopic results indicated the surface of MBs became surrounded by red fluorescence, demonstrating DiI-labled MBs were successfully synthesized.2. HPLC and mass spectrum analysis:The molecular weight of the synthesized peptide was1559.86Da, consistent with the theoretic molecular mass of1560Da. The synthesized peptide purity was about97.8%.3. Characterization of biotinylated H-F-Tat-P NPs:The mean diameter of biotinylated H-F-Tat-P NPs was around120±7nm and zeta potential was approximately-35±4mV. The morphology of biotinlyated H-F-Tat-P NPs displayed an approximately spherical shape as determined by TEM. The content of paclitaxel was about8.84%as determined by UV method.4. Characterization of biotinylated NPs-loaded MBs:The microscopic results indicated the surface of NPs-loaded MBs became surrounded by yellow fluorescence, demonstrating Oregon green labeled NPs were successfully attached to the surface of Dil-labled MBs.5. Results of cellular uptake experiments5.1. The optimization of ultrasonic irradiation conditionsUnder irradiation intensity1.0W/cm2and irradiation time40s, MBs were destructed to perforate cell membranes thereby promoting higher amount of NPs (green fluorescence) centralized on nucleus (staining by Hochest33342) in MDA-MB-231cells. Under irradiation intensity1.5W/cm2and irradiation time40s, the fluorescent signal of NPs could only be strengthened in cytoplasm and partial nucleus for A549cells. Under other ultrasonic conditions, it was difficult to observe NPs signal in both cells.5.2. Quantitative analysis of cellular uptake of NPs and NPs-loaded MBs with and without US by flow cytometry:the extend of celluar uptake for A549cell lines was:NPs-loaded MBs with US exposure>H-F-Tat-P NPs> NPs-loaded MBs without US exposure. For MDA-MB-231cells:NPs-loaded MBs with US exposure>H-F-Tat-P NPs> NPs-loaded MBs without US exposure.5.3. Qualitative analysis of cellular Uptake of H-F-Tat-P NPs by confocal laser scanning microscopy:the fluorescent signal of NPs in MDA-MB-231cells was higher than that of A549cells under the same conditions. For both two cell lines, ultrasonic irradiation can promote cellular uptake of NPs.6. Cell Viability AssayThe results of cell viability for both two cell lines were:MBs+US> free paclitaxel> H-F-Tat-P NPs>NPs-loaded MBs+US.(P<0.05) Conclusions:1. The optimal ultrasonic irradiation conditions for MDA-MB-231cells, irradiation intensity1.0W/cm2, and irradiation time40s; and for A549cells, irradiation intensity1.5W/cm2, and irradiation time40s.2. The FR-mediated endocytosis, Tat-mediated transmembrane function and ultrasound-mediated effect together contributed to the enhancement of cellular uptake of nanoparitlcles. |
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