Da-Yan Yang, Qi-Qing Chen, Ting-Ting Zhong, Min Zhang, Yan Chen, Lin Xie, Xiang-Xiang Jing, Ling Lin?, Ming-Hua Wang

1. Hainan general hospital (Affiliated Hainan hospital of Hainan medical college), Haikou 570311, Hainan province, China 2. The second affiliated hospital of Hainan medical college, Haikou 570311, Hainan province, China

Keywords:Folic acid Targeting Cisplatin Ultrasound

ABSTRACT Objective: To prepare a encapsulated liquid fluorocarbon,multimodal nanoscale ultrasonic molecular probes capable of carrying Cisplatin(CDDP)、Fe3O4 and folic acid molecular targeting, study on its basic characteristics and in vitro targeting ability. Methods: The PLGAFa、Fe3O4、CDDP and PFP were dissolved in organic solvents in a certain proportion after the folic acid (Fa) molecule which in the surFace of PLGA was attached to polyethylene glycol imide, prepare nanoscale targeted multimodal ultrasound contrast agent PLGA-Fa/Fe3O4/CDDP/PFP,containing Fe3O4、CDDP、PFP by double creaming methods,the basic properties of it,encapsulation efficiency and drug loading of nanometer particle were observed and measured to optimize the optimal dosage of CDDP; the PFFCP and PFCP were acted on in vitro human nasopharyngeal carcinoma (NPC) cell line HNE-1 (high expression of Fa receptor),observe and compare its targeting. Results: Nanoscale targeted multimodal ultrasound contrast agentPLGA-Fa/Fe3O4/CDDP/PFP containing Fe3O4、CDDP、PFP was successfully prepared, the size of the nanometer particle is uniform, the shape is round, the dispersion is good, the diameter distribution is 204nm, and the average electric potential is -15Mv. The nanometer particle could bind to human NPC cell line HNE-1 cells with high expression of Fa receptor, but the nontarget nanometer particle binding of human NPC cell line HNE-1 cells are not obvious. Conclusion: In this study, nanoscale targeted multimodal ultrasound contrast agent PLGA-Fa/Fe3O4/CDDP/PFP containingFe3O4、CDDP、PFP was successfully prepared, and the optimal dose of CDDP was optimized. The nanoparticle have obvious targeting to human NPC cell line HNE-1 cells.

1. Introduction

Hainan province has a high incidence of nasopharyngeal carcinomaand ,NPC in the middle and late stage and not sensitive to radiation have poor therapeutic effect[1].It is urgent to provide an efficient and visual diagnosis and treatment method for NPC patients [2-3].Targeted drug delivery of tumor is a research hotspot and has a very good application prospect.Folic acid (folic acid Fa) receptor is one of the research hotspots of many tumor targets [4-6]. The study of multifunctional multimodal ultrasound contrast agent provides a new treatment method for tumor molecular targeting drug delivery system, which is expected to realize visualized precise targeted therapy for tumors [7-9].This study aims to develop a novel multimodal, multifunctional nanoscale molecular probe with Fa molecular targeting, which can be loaded with Cisplatin CDDP chemotherapy drugs and superparamagnetic Fe3O4 nanoparticles, and coated with liquid perfluoropentane PFP that can undergo liquid-gas phase change.

2. Preparation and characterization of multifunctional targeting nanometer targeting probe loaded with cisplatin

2.1 Materials and Methods

Main reagents and equipment: polylactic-co-glycolic acid(PLGA),50:50,25 thousands,

Jinan daigang biological engineering co. LTD; pol vinyl alcohol, PVA, USA Sigma; cisplatin(CDDP) USA Sigma; folic acid(Fa) USA Sigma;PFP, J&K Chemicals;SONICS ultrasonic acoustic vibrator, ZNCL - BS intelligent digital display magnetic agitator, Eppendorf 5804 (R) multi-functional high speed centrifuge, Malvern laser particle size meter (3000SSA, Zetasizer, USA), scanning electron microscopy (Hitachi, Japan), transmission electron microscopy (TECNAI - 10, PHILIPS).

2.2 Preparation of Fa-PLGA targeting membrane material

With carbon ethyleneimine method, using polyethylene glycol (PEG) at the end of the NH3 and PLGA is activated at the end of the COOH base reaction, forming amide bond, connected the PEG to PLGA first, then carbon ethyleneimine method is used to connect the folic acid Fa to PEG - PLGA, preparation, targeting membrane film recycling target material to take medicine and other decorate, can increase the rate of targeted nanoparticles into the ball, increase the utilization rate of folic acid.

2.3 Preparation of multi-mode nanoscale ultrasound contrast agent with CDDP.

50 mg plga-fa was dissolved in 2ml dichloromethane and 80ul oil was added to acidify Fe3O4.150UL PFP and 150UL CDDP solution with concentration of 40mg/ml were added to the dichloromethane solution with plga-fa.(3) with the ultrasonic acoustic vibrator, pulse wave oscillation for 2.5min, 5 seconds ON/5 seconds off, the energy output is 45%w, can be obtained white emulsion (w/o particles);(4) 5ml4%PVA was added, and then the ultrasonic vibrator was used again, pulse vibration was 2.5min, 5 seconds ON/5 seconds off, the energy output was 45%w (w/o/w particles), and then 5ml of 2% isopropanol was added.(5) under the condition of ice bath, the magnetic stirrer is stirred for 5 h (10-15r/m) to fully volatilize dichloromethane;6) double steam water high-speed low-temperature centrifugal washing 3-5 times (10000r/min, 5min), the collection of plga-fa /Fe3O4/CDDP/PFP (PFFCP) nanoparticles.Non-targeted nanoparticles PLGA/Fe3O4/CDDP/PFP(PFCP) were prepared by the same method mentioned above, and finally the targeted PFFCP and non-targeted PFCP particles were stored in a standby refrigerator at 4℃.

2.4 Potential, particle size and other characterization of PFFCP multifunctional nanoparticles

The appearance, morphology, structure and dispersion were observed by optical microscope, scanning electron microscope and transmission electron microscope.The mean particle size and surface potential were measured by Marvin particle size measuring instrument and potential measuring instrument.

2.5 The encapsulation rate and drug loading capacity of PFFCP nanoparticles were determined by HPLC

The supernatant in the process of preparing PFFCP nanoparticles was collected, and the content of CDDP in the supernatant was measured by high performance liquid chromatography. The formula was used:Drug loading rate = (total dosage - CDDP amount in supernatant)/total amount of nanoparticles, encapsulation rate = (total dosage - CDDP amount in supernatant)/total dosage, and the drug loading and encapsulation rate of CDDP of PFFCP were calculated.

2.6 The continuous target rate of nanoparticles was observed by fluorescence confocal microscopy

During the preparation of nanoparticles, DiI red fluorescent dye was added to the oil phase in the first step to make the PLGA display red fluorescence.After the preparation of nanoparticles, using mice with folic acid resistant IgG nanoparticles in 1:500 scale, under the condition of ice bath, placed in the table, at a speed of 120 r/m, incubation 4 hours together, centrifugal precipitation heavy suspension, add FITC in proportion of 1:100, ice bath conditions, placed in the table, at a speed of 120 r/m, continue incubation for 1 hour, centrifugal, washing, collection of nanoparticles, the green fluorescence confocal fluorescence microscope of folic acid and red fluorescence coincidence of membrane materials, and even the target to observe nanoparticles.

2.7 Culture of HEN1 cells from human nasopharyngeal carcinoma

Culture conditions: the cells were cultured in DMEM high sugar medium containing 10% high-quality fetal bovine serum and 1% penicillin-streptomycin double antibody. The cells were subcultured once after 2-3d liquid exchange. The cells were subcultured at 80% fusion degree.

2.8 In vitro target finding

HEN-1 in the logarithmic growth stage was inoculated into a sixwell plate at a density of 1×105/ well, and then cultured for 24 hours. The samples were divided into two groups, with three repores in each group. Targeted nanoparticles and non-targeted nanoparticles were added with 1ml of medium mixture at a concentration of 100ug/ml.

3. Results

3.1 Experiment results

Under the light microscope, the nanoparticles were uniform in size and evenly dispersed (FIG 1). Under the fluorescence microscope, the nanoparticles after DiI staining showed red fluorescence with good staining effect (FIG 2).Transmission electron microscopy (tem) results showed that PFFCP nanoparticles were spherical and the shells had a large number of high-density black material distribution, which indicated that Fe3O4 successfully loaded the PLGA nanoparticles shells (FIG 3).The average particle size was 204.8±60.64nm and the average surface potential was -15.2±5.04MV (FIG 4-5).

FIG. 1 ordinary light lens ×600;FIG. 2 fluorescence microscope x200;FIG. 3 transmission electron microscopy (tem), the gray circle is a nanosphere, and the black particles on it are Fe3O4.FIG. 4 average particle size detected by Marvin particle size analyzer was 204.8±60.64nm.FIG. 5 average potential of -15.2±5.04MV

3.2 Confocal observation of the target effect of nanoparticles

The red color is ddi-stained PLGA, and the green color is the connection between FITC and folic acid on the surface of the nanoparticles. The fusion of the two is yellow, that is, the yellow surface is connected with the nanoparticles with folic acid. According to the confocal display results, it can be seen that the tami grains have a high continuous target rate (FIG 6-8).

3.3 Results of encapsulation rate and drug loading

Encapsulation rate and drug loading of PLGA/CDDP nanoparticles were determined by HPLC.In the preparation process, the total dosage was 6mg, the detected concentration of cisplatin in the supernatant for testing was 4ug/ml, the total amount of supernatant was 15ml, and the content of CDDP in the supernatant was 60ug. According to the formula, the encapsulation rate was 99%, the drug loading was 5.94mg, and the prepared nanoparticles were 2ml in total. According to the formula, the drug loading rate per ml was 2.97mg/ml.

3.4 In vitro target finding results

After co-incubation of targeted nanoparticles and untargeted nanoparticles with hne-1 cells with high expression of folic acid receptor for 2 hours, the targeted nanoparticles adhered to the cell periphery more than the untargeted nanoparticles, showing obvious targeting (FIG 9-10).

FIG 6 shows the PLGA dyed by DiI with red fluorescence;FIG 7 shows FITC dyed FA with green fluorescence;FIG. 8 shows the coincidence of the two, showing yellow fluorescence;FIG 9 shows the adhesion of non-targeted nanoparticles to cells.FIG 10 shows the adhesion of targeted nanoparticles to cells.

4. Conclusion

Success of this study was prepared by a nanoscale carrying Fe3O4, CDDP, PFP targeted mode ultrasound contrast agent Fa-PLGA/Fe3O4/CDDP/PFP, the good dispersion, uniform size, high drug loadings, the nanoparticles of NPC cell lines Alexandra bohne - 1 has obvious target cells.Its successful preparation for the next step the accurate treatment of tumor cells laid a good foundation.

5. Discussion

During the experiment, the target connection mode on the surface of nanoparticles was adjusted. The traditional target connection mode of targeted contrast agents was to prepare drug-loaded nanoparticles with a single membrane material and then connect the target to the surface of nanoparticles, which had a low pellet formation rate.In our improved method, PEG was firstly connected to PLGA by c-ethylamine method, and then folic acid Fa was connected to peg-plga by c-ethylamine method to prepare targeted membrane materials, and then targeted membrane materials were used for drug loading and other modification, which could improve the pellet-forming rate of targeted nanoparticles and increase the utilization rate of folic acid [10].In the preparation process of nanoparticles, it was found that in the double-emulsion preparation process, the second emulsification method was used to replace the high-speed homogenizer with ultrasonic vibrator, and the particle size of the obtained nanoparticles would be significantly reduced and the homogeneity of nanoparticles would be high [11].Wrapped in nanoparticles in liquid fluorocarbon, commonly used have PFP and perfluorinated hexane (perfluorohexanes PFH) two [12], the boiling point to 29 degrees Celsius, the latter is 56 degrees Celsius [13], but the liquid fluorocarbon package into the PLGA membrane, itself is the boiling point will raise, the former about 50 degrees, which needs more than 70 degrees [14, 15], experimental conditions combined with the body, we chose the PFP as the kernel of liquid fluorocarbon.After a series of optimizations, we successfully prepared the nanoscale ultrasound contrast agent with phase transition and targeted drug delivery, which laid a foundation for the follow-up in vitro and in vivo experiments.