Facile Construction of Chloroquine Containing PLGA-Based pDNA

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Facile construction of chloroquine containing PLGA-based pDNA delivery system for efficient tumor and pancreatitis targeting in vitro and in vivo Chengli Yang, Tingting Hu, Hua Cao, Lijing Zhang, Pengxiang Zhou, Gu He, Xiangrong Song, Aiping Tong, Gang Guo, Fan Yang, Xiaoning Zhang, Zhiyong Qian, Xiaorong Qi, Liangxue Zhou, and Yu Zheng Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00155 • Publication Date (Web): 08 May 2015 Downloaded from http://pubs.acs.org on May 13, 2015

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Molecular Pharmaceutics

Facile construction of chloroquine containing PLGA-based pDNA delivery system for efficient tumor and pancreatitis targeting in vitro and in vivo

Chengli Yanga#, Tingting Hua#, Hua Caoa, Lijing Zhanga, Pengxiang Zhoua, Gu Hea, Xiangrong Songa, Aiping Tonga, Gang Guoa, Fan Yanga,b, Xiaoning Zhangc, Zhiyong Qiana, Xiaorong Qib, Liangxue Zhoua,d*, Yu Zhenga* 1,17#, Section 3, Ren Min Nan Lu, Chengdu, Sichuan 610041, P.R.China. a, State Key Laboratory of Biotherapy/Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University, Chengdu,610041, P.R.China. Fax: +86 2885164060.Tel: +86 28 85503817 E-mail: [email protected] b, Department of Gynecology, West China Second University Hospital, Sichuan University, Chengdu,610041, P.R.China c, Laboratory of Pharmaceutics, School of Medicine, Tsinghua University, Beijing, 100084, P.R.China d, Department of Cerebral Surgery, West China Hospital, Sichuan University, Chengdu,610041, P.R.China

#

The first two authors contributed equally to the work.

*E-mail:[email protected] (Y. Zheng) and liangxue [email protected] (L.X.Zhou)

Abstract Chloroquine diphosphate (CQ) was ingeniously to take place of phosphate salt in traditional calcium phosphate coprecipitation method for pDNA transfection. With multiple roles of CQ in the novel Ca-CQ-pDNA complex including pDNA compaction and assistance in lysosome escape, the transfection efficiency of the pDNA was significantly increased relative to the traditional method. CQ did not intercalate into the DNA double helix as free CQ did, which was probably ascribed to the prior mixing of the pDNA with high concentration of calcium chloride. In order to construct efficacious vector for in vivo gene delivery, Ca-CQ-pDNA-PLGA-NPs was designed and prepared. With entrapment efficiency, particle size and pDNA integrity as screening conditions, the optimal prescription was obtained and CaPi-pDNA-PLGA-NPs made with classic calcium phosphate coprecipitation method after optimization was also prepared as control to systematically study the role of CQ in the novel vector. Physical characters of the vectors were comprehensively studied using TEM, DSC and XRD.

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The safety of the vector both in vitro and in vivo was evaluated using MTT, hemolysis test and histological sections. The Ca-CQ-pDNA-PLGA-NPs dramatically enhanced the gene tranfection efficiency in Human Embryonic kidney HEK293 cells compared with the CaPi-pDNA-PLGA-NPs and presented an increasing gene transfection for up 144h. The relative fast release of the CQ compared with pDNA from the nanoparticles was responsive for the increased transfection. The Did-labeled-Ca-CQ-pDNA-PLGA-NPs exhibited excellent tumor targeting efficiency and sustained circulation time in CT26 mouse model. The Ca-CQ-pDNA-PLGA-NP loaded with the plasmid pVITRO2 expressing mSurvivin-T34A protein excerted 70% tumor inhibition rate, which was partially ascribed to CQ. The Ca-CQ-pDNA-PLGA-NPs showed high targeting efficiency in C57 acute pancreatitis model. In all, the Ca-CQ-pDNA-PLGA-NP was a promising candidate for targeted gene delivery to both tumor and pancreatitis. Keywords:

Chloroquine

containing

PLGA-based

pDNA

delivery

system,Enhanced

lysosome

escape,Ca-CQ-pDNA-PLGA-NP,Tumor targeting,Pancreatitis targeting Introduction It has been the key issue in gene therapy to construct nonviral gene delivery system with low immunogenicity, high efficiency and great biodegradability. The PLGA-based gene delivery system has been widely used for drug delivery system due to excellent biocompatibility and biodegradability. PLGA is hydrolyzed into two monomers, lactide and glycolide, which will enter the tricarboxylic acid cycle and are easily metabolized

1，2

PLGA is negatively charged under physiological condition, so it can effectively avoid hemolysis which is often encountered by other cationic gene delivery carrier. And pDNA is always immobilized within a core encapsulated by the PLGA diffusional barrier, which will effectively avoid enzymatic degradation during transport 3. Apart from PLGA, a cationic material is needed to bind pDNA through electrostatic interaction. In our previous study 3, the biodegradable calcium phosphate (CaPi) has been used, and the obtained nanoparticles were named CaPi-pDNA-PLGA-NPs. However, the tumor targeting efficiency of the vector was not very ideal after intravenous administration and intratumoral administration was adopted for antitumor therapy4. In purpose of enhancing tumor targeting of nanoparticle, cationic polymer like PEI was usually used to facilitate the lysosomal escape of the nanoparticle5-7. Because of concerns about the toxicity of the cationic polymers, the small molecule drug chloroquine diphosphate (CQ) was used in this study. CQ has been widely used to assist lysosome escape of gene through the mechanisms comprising of destruction of the endosome mature and inhibition of the lysosomal enzyme under elevated lysosomal 、 pH due to “proton sponge effect” of CQ8-11.


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As we know that the CaPi-pDNA complexes were formed through the electrostatic interaction between calcium ion and phosphate groups of pDNA and through the compaction of the pDNA molecules12, we hypothesized that the CQ possessing phosphate radicals might replace disodium hydrogen phosphate in traditional calcium phosphate coprecipitation method and form Ca-CQ-pDNA complex for gene transfection. Besides, the Ca-CQ-pDNA complexes might be encapsulated into the PLGA nanoparticles named Ca-CQ-pDNA-PLGA-NP. The synchronous release of the CQ with the pDNA from the nanoparticles after endocytosis might increase gene transfection, enhance nanoparticle escape from lysosome and achieve tumor targeting through intravenous nanoparticle delivery. CQ has firstly been discovered to treat malaria. Recently, more pharmacological actions of CQ including antitumor, vessel normalization effect and immunosuppressive effect, etc have been reported and some relevant clinical trials have also been covered13

，14

. So, CQ might exert pharmacological effect in the

course of therapy and exploit synergies with the therapeutic gene. In all, there are three roles that CQ might play in the novel Ca-CQ-pDNA-PLGA-NPs. Firstly, CQ provides phosphate radical to compact pDNA. Secondly, CQ might increase tumor targeting of the nanoparticle by assisting lysosomal escape. Thirdly, CQ might show synergistic anti-cancer and anti-inflammatory effect with the therapeutic gene. As shown in the schematic diagram (Fig.1), the Ca-CQ-pDNA complexes were encapsulated in the aqueous phase. Water-in-oil-in-water (w/o/w) double emulsion solvent evaporation method was used to prepare the Ca-CQ-pDNA-PLGA-NPs. PLGA nanoparticles have been used for drug delivery to macrophages and for therapy of inflammatory disease15. To our best of knowledge, the PLGA-based drug delivery nanoparticles have not been used for targeted delivery to pancreatitis. In this study, the novel PLGA-based gene delivery system will be studied for its targeting efficiency in mouse tumor model and mouse acute pancreatitis (AP) model. Gene therapy for pancreatitis has been reported to be more efficient than pharmacologic therapy to exert anti-inflammatory effect16-18. Recently, gene therapy for pancreatitis pain has exhibited significant analgesia for the duration of the transgene expression (approximately 4-6 weeks) 17. We hypothesized that the novel PLGA-based system would exert ideal therapeutic effect in mouse tumor model and pancreatitis mouse model if it exhibits excellent targeting efficiency. Fig.1 Schematic diagram for preparation of the Ca-CQ-pDNA-PLGA-NPs and its mechanism of pDNA intracellular delivery. (a) Entery of the nanoparticle into endosome by endocytosis; (b) Progressive decline of pH in lysosome; (c) Prevention of the pH decrease in lysosome due to ‘the proton sponge effect’; (d) Rupture of lysosome due to the CQ and escape of the pDNA from the lysosome after that; (e ) Entery of the released pDNA



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into nucleus for active transcription.

2 Materials and methods 2.1 Materials PLGA 50:50 (lactide:glycoside, MW=20 kDa) was purchased from Shandong Institute of Medical Instruments(Shan Dong, China). PVA (MW: 30,000–50,000, HD: 87%) and Hoechst33258 reagent were both purchased from Sigma–Aldrich. Red Fluorescence Protein Plasmid DNA (RFP) and pVITRO2 plasmid DNA were purchased from Invitrogen Corporation (San Diego, California). The plasmid pVITRO2 expressing mSurvivin-T34A protein was constructed in our laboratory as described previously19. The recombinant plasmid was extracted from cultured Escherichia coli with Qiagen EndoFree Plasmid Giga Kit (Santa Clara, CA). Both chloroquine diphosphate (CQ) and 4′,6-diamidino-2-phenylma-Aindole (DAPI) was purchased from J&K Scientific,Ltd (Bei Jing, China). Coumarin-6 (C6) was purchased from TCI Development Co, Ltd (Shang Hai,China). 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate (Did) was purchased from Biotium,Inc (Hayward, CA). Lyso-Tracker, a lysosomotropic probe was purchased from Invitrogen (Shanghai, China). Human embryonic kidney (HEK293) and mouse colon carcinoma cell (CT26) lines were obtained from American Type Culture Collection (ATCC, Rockville, M).All other chemicals used were analytical grade reagent. 2.2 Cell Culture The HEK293 and the CT26 cells were both maintained in Dulbecco's minimum essential medium(DMEM, Gibico, USA) supplemented with 10% fetal bovine serum, and 1% penicillin/streptomycin and cultured at 37°C in a 5% CO2 incubator. 2.3 Preparation and characterization of the Ca-CQ-pDNA complexes 2.3.1Preparation of the Ca-CQ-pDNA complex The calcium precursor solution was comprised of pDNA and CaCl2 (2.5M). The CQ precursor solution with varied Ca/CQ ratios of125, 250, 500, 1000, 2500 were prepared. Then, the Ca-CQ-pDNA complex was prepared by mixing the calcium precursor solution with the CQ precursor solution followed by standing at room temperature for 20 minutes before use. The calcium phosphate-pDNA (CaP-pDNA) complexes prepared as following were used as control. The calcium precursor solution was comprised of pDNA and CaCl2 (250mM). The disodium hydrogen phosphate precursor solution (1.5mM) was prepared. Then, the CaP-pDNA complex was prepared by mixing the calcium


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precursor solution with the phosphate precursor solution followed by standing at room temperature for 20 minutes before use. 2.3.2 Electrophoresis Ca-CQ-pDNA complex were applied to a 0.8% agarose gel electrophoresis in TBE buffer (89mM Tris, 89mM Boric Acid, and 2mM EDTA, pH8.4) containing 0.6 μg ml-1 ethidium bromide. Electrophoresis (Sunrise 96, Labrepco, USA) was carried out at a constant voltage of 120V for 20min. Images under fluorescent light were captured by a gel documentation system (Bio-Rad Laboratories, Inc, Hercules, CA). 2.3.3 Transfection of the Ca-CQ-pDNA complexes in vitro in HEK293 and CT26 The cells were seeded into 6-well cell culture plates at a cell concentration of 1 × 105 cell per well a day before transfection. After 24h, the old media were removed and replaced by fresh medium with the Ca-CQ-pDNA complexs (2 μg pDNA per well). After 48 h of transfection, the cells were imaged using an Olympus IX-70-fluorescence microscope (Tokyo,Japan) with a dichroic filter set (ex 555 nm, em 584 nm). After that, the cells were harvested and the transfection efficiency was analyzed by a FACS Calibur ﬂow cytometer (FACS Canto II, BD Biosciences, San Jose, CA). Three independent experiments were performed. 2.4 Preparation and optimization of the Ca-CQ-pDNA-PLGA-NP prescription 2.4.1 Nanoparticle preparation The Ca-CQ-pDNA-PLGA-NPs were prepared by a water-in-oil-in-water (w/o/w) double emulsion solvent evaporation method. PLGA (7.5mg) was dissolved in 0.75ml of dichloromethane-ethyl acetate mixed (DCM–EA) (1/1, v/v) as the organic phase. The inner aqueous phase was the mixture of 125ul of calcium precursor solution and 125ul of CQ precursor solution. The calcium precursor solution was comprised of different amount pDNA and CaCl2 with different concentrations. The CQ precursor solution was comprised of 0.28M NaCl, 10mM KCl, 50mM dextrose, 40mM HEPES and CQ with different concentrations. Then, the organic phase was emulsified with the aqueous phase by sonication using a microtip probe sonicator (Tekmar Sonic Disruptor TM300, Mason, Ohio) at certain amplitude for 60s in ice bath. The primary emulsion was then added into 3ml PVA solution with different concentrations and the mixture was sonicated for 60s at certain amplitude in ice bath. The organic phase in the mixture was rapidly removed by evaporation under reduced pressure at 37°C leaving behind the colloidal suspension of the PLGA nanoparticles in water. The nanoparticle solution was ultracentrifuged at 4°C at a speed of 13,300rpm for 90min (Eppendorf, Germany). The supernatant was discarded and the nanoparticles in the precipitate were redispersed in Milli-Q water until further use. The CaPi-pDNA-PLGA-NPs were prepared referring to the previously established method3. The main



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difference between the Ca-CQ-pDNA-PLGA-NPs and the CaPi-pDNA-PLGA-NPs was that CQ was used to substitute disodium hydrogen phosphate to condense pDNA. The fluorescent dye-labeled nanoparticles were prepared as described above with the dye addition in the organic phase containing PLGA. 2.4.2 Particle size and zeta potential The nanoparticle size distribution and zeta potential were measured by a laser particle size analyzer (Malvern Nano-ZS 90, UK) utilizing dynamic light scattering (DLS) technique. Samples in triplicate from the prepared suspensions were diluted in Milli-Q water and placed in measurement cell for analysis. 2.4.3 Determination of pDNA and CQ entrapment efficiencies The encapsulation efficiency was calculated by dividing the pDNA or CQ amount in the particle fraction by the amount of pDNA or CQ added initially. The amount of pDNA or CQ in the particle fraction was indirectly calculated by subtracting the amount of unencapsulated pDNA or CQ in the supernatant from the amount of pDNA or CQ added initially. The amount of pDNA was quantified by pDNA fluorometric assay using Hoechst dye 33258 as described in previous study20. Briefly, 200ul of the supernatant was mixed with 3.8ml 0.15 μg ml-1 Hoechst 33258 solution.The fluorescence intensity was determined on a fluorescence spectrophotometer at an excitation wavelength of 356nm and an emission wavelength of 465nm (LS35, Perkin-Elmer, Norwalk, CT). The amount of CQ in the supernatant was examined with UV spectrophotometry at a maximum absorption wavelength of 343nm (Lambda 35, Perkin-Elmer, Norwalk, CT). 2.4.4 pDNA integrity The protection effect of the nanoparticles on pDNA was evaluated by gel electrophoresis. Dichloromethane (40ul) was added to the precipitate of the nanoparticles after ultracentrifugation to dissolve the nanoparticles by vortexing for 10 seconds. TBE buffer with 2% (w/v) heparin sodium was added to set the pDNA free. pDNA samples recovered from the nanoparticles were applied to a 0.8% agarose gel electrophoresis in TBE buffer containing 0.6 μg ml-1 ethidium bromide. Electrophoresis was carried out at a constant voltage of 120V for 20min. Images under fluorescent light were captured by a gel documentation system. Dichloromethane was used to dissolve the nanoparticles and the heparin was used to replace the combined pDNA. The method can extract pDNA from the nanoparticles completely. The pDNA amount did not influence the pDNA integrity. 2.5 XRD analysis The Ca-CQ-pDNA-PLGA-NPs, Ca-CQ-pDNA (prepared as described in section 2.3.1 with a CQ precursor


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solution of 2.0mM), blank PLGA nanoparticles (prepared as described in section 2.4.1 except that calcium precursor, CQ precursor and pDNA were replaced by Milli-Q water). All the samples were lyophilized before analysis without addition of the lyoprotectants. X-ray diffractometer (X’Pert Pro, Philips, Netherlands) was utilized to study the crystalline phases of the samples. XRD studies were performed by exposing samples to CuKa radiation (40 kV, 20 mA). Lyophilized samples used for XRD analysis were Ca-CQ-pDNA, pDNA-PLGA-NPs and Ca-CQ-pDNA-PLGA-NPs. Film samples were scanned continuously over an angular range between 0° and 70°(2θ) with a step size of 0.02°. The scan step time was 1.2°/min. 2.6 Differential scanning calorimetry (DSC) analysis The thermal properties of the Ca-CQ-pDNA, the blank PLGA nanoparticles and the Ca-CQ-pDNA-PLGA-NPs were also analyzed by DSC (NETSCZ 204, Germany). DSC thermograms were acquired by heating the samples in a sealed standard aluminum pans (TA instrument, New Castle, Delaware, USA) at a heating rate of 10°C/min over a temperature range of 10°C to 400°C. 2.7 Transmission electron microscopy (TEM) Morphology of the Ca-CQ-pDNA-PLGA-NPs were analyzed on a TEM (H-600, Hitachi, Japan) following negative staining using the phosphotungstic acid. 2.8 Cytotoxicity evaluation by MTT assay Cytotoxicity

of

the

Ca-CQ-pDNA-PLGA-NPs

was

determined

by

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

5-diphenyltetrazolium bromide) MTT assay in HEK293 cells. HEK293 cells were seeded in 96-well plates at 2 × 103 cells per well followed by culture for 24h. The nanoparticles with different final concentrations in the media (0.01, 0.25, 0.5, 1, 2, 4, 6, 8, 12mg ml-1) of the Ca-CQ-pDNA-PLGA-NPs were used to treat the cells for 48h. 20µl MTT solution (5mg ml-1 in PBS, pH7.4) was added to the treated cells and incubated for 4h. The formazan crystals formed were dissolved using 150µl per well Dimethyl sulfoxide（DMSO) and absorbance was read at 570nm on a microplate reader. Cell viability was determined as a percentage of the negative control (untreated cells). 2.9 Hemolysis Healthy erythrocytes were washed 3 times with saline water and suspended in the same buffer. The Ca-CQ-pDNA-PLGA-NPs were added to the suspension of erythrocytes to the final concentrations of 1.6, 3.2, 4.8, 6.4, 8mg ml-1 for 3h at 37 °C. At the same time, the Healthy erythrocytes were treated with distilled water and saline water to serve as a positive and a negative control, respectively. All the mixture was centrifuged at



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1500rpm for 10min. The release of hemoglobin in the supernatant was determined by UV spectrophotometry at 545nm. The sample with a hemolysis less than 5% stands for a good hemocompatibility and non-toxicity against erythrocytes membranes as described elsewhere. 2.10 H&E Staining For histopathological studies, the Ca-CQ-pDNA-PLGA-NPs were administered to balb/c mice at a concentration of 100mg kg-1 as i.v. injection through the tail vein. The mice were sacrificed and excised tissue samples were fixed in 10% phosphate buffered formalin and embedded in paraffin, then cut in 5um thick paraffin sections and subsequently placed on glass slide. The slides were stained with hematoxylin and eosin (H&E) according to the method of previous study21. The histopathological alterations were observed and imaged with a light microscope (OLYMPUS BX43, Tokyo, Japan). 2.11 pDNA and CQ release from the nanoparticles in vitro pDNA release from the nanoparticles was studied in phosphate buffered saline (PBS) at pH4.5, pH6.8 and pH7.4. Approximately 7.5mg of the Ca-CQ-pDNA-PLGA-NPs were suspended in 1ml of PBS containing 0.02% sodium azide as release medium at 37°C on a horizontal shaker at 100rpm. Samples were taken at 0.5h, 2h, 4h, 6h,18h, 1d, 2d, 4d, 7d, 2w, 3w, 4w. At each time point, the particle suspensions were ultracentrifuged at 13,300rpm, and the supernatant was completely with drawn.The concentrations of the pDNA released in the supernatant were determined by Hoechst 33258 staining assay as described in section2.4.3. After that, 1ml of the fresh release medium was added to resuspend the particles. 2.12 Cellular uptake in vitro CT26 cells at an initial density of 5 × 104 cells per well were plated in 24-well plates. After 24h of cell culture, the medium was replaced by fresh medium containing the C6 labeled nanoparticles with the final C6 concentration of 25ng ml-1 in the media. After incubation for 0.5, 2, 6,12h with CT26 cells, the media were removed and the lysosomal staining was performed using Lyso-Tracker. After staining, the media were removed and cells were washed three times with cold PBS. Then, freshly prepared 4% (w/v) paraformaldehyde (500µL) was added into each well, and cells were fixed for 15min and washed three times using PBS (10mM, pH 7.4). Nuclei were counter-stained with DAPI at final concentration of 1μg mL-1. Finally, cells were observed under a florescent microscope and imaged using an OLYMPUS BX43 fluorescence microscope. 2.13 Transfection of the nanoparticles in vitro 2 × 105 CT26 cells were plated into 6-well plate. After 24h, The old medium was discarded and replaced by fresh medium with the Ca-CQ-pDNA-PLGA-NPs nanoparticles (4μg pDNA per well). The nanoparticle


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containing medium was replaced with fresh medium at 24h. After various time intervals (24, 48, 72, 96, 144h), cells were observed under a fluorescent microscope and imaged using an Olympus IX-70-fluorescence microscope with a dichroic filter set (ex 555nm, em 584nm). Three independent experiments were conducted. 2.14 In-vivo imaging of tumor-bearing mice after treatment of the Ca-CQ-pDNA-PLGA-NPs To establish the subcutaneous tumor model, exponentially growing CT26 cells were suspended in serum and antibiotic-free RPMI-1640 medium to a final concentration of 1×107cells/mL. Cell suspensions (1×106cells/100µL) were injected into the subcutaneous flanks of 6-week-old balb/c male mice near the hind limb. Tumor growth was measured with a caliper, and tumor volume (mm3) was calculated as V=0.5×length×width2. C6 labeled Ca-CQ-pDNA-PLGA-NPs were administered to the mice at 0.5mg kg-1 of C6 as a i.v. injection through the tail vein. Mice were sacrificed and all major organs including heart, live, spleen, lung, kidney and tumors were removed and imaged at 4, 12, 24, 48h postinjection by a in vivo fluoresence imaging system(Quick View 3000 Bio-Real,Austria).The fluorescence filters were set at ex 474nm and em 525nm. Meanwhile, for in vivo fluorescence imaging, tumor-bearing mice were given saline water, Did labeled CaPi-pDNA-PLGA-NPs, Did labeled Ca-CQ-pDNA-PLGA-NPs through tail vein injection at 0.2mg kg-1 of Did. At the time point of 4h postinjection, the mice were anesthetized for in vivo fluorescence imaging by a Quick View 3000 Bio-Real. The fluorescence filters were set at ex 655nm and em 714nm. All experimental protocols and animal handling procedures were approved by the Institutional Animal Care and Use Committee of Sichuan University.

2.15 Antitumor activity of ms-Ca-CQ-pDNA-PLGA-NPs in vivo

It was well known that survivin is a prognostic indicator for poor survival in malignancy22. Dominant negative mutant to target survivin has been proved to be a promising approach for cancer therapy19. The dominant negative mutant (survivin Thr34 → Ala) constructed previously in our research group named pVITRO2 expressing mSurvivin-T34A was used as a model gene to evaluate the tumor therapy effect of the vector developed in this study. pVITRO2 loaded vector was named null-Ca-CQ-pDNA-PLGA-NPs. pVITRO2 expressing mSurvivin-T34A loaded vector was named ms-Ca-CQ-pDNA-PLGA-NPs. Tumors were inoculation as described in section 2.14. Mice were randomly divided into 4 groups (5 mice per



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group) as the the tumor grew to an average of 100cm3 (V=0.5×length×width2). The mice were treated with saline water, the vector itself, null-Ca-CQ-pDNA-PLGA-NPs, ms-Ca-CQ-pDNA-PLGA-NPs through the tail vein (5µg pDNA/mouse) once every two days for 9 times (days 6 to 22). On day 24 (2 days after treatment), all mice were sacrificed. Tumor net weight of each mouse was measured. During the assay, the tumor size was determined by caliper measurement of the largest and smallest superficial diameters every two days. Immunohistofluorescence analysis of microvessel formation were performed with goat anti-mouse CD31 anti-body (abcam, MA, USA) using the SABC immunohistochemistal method. Sections were observed under a fluorescent microscope and imaged using an Olympus IX-70-fluorescence microscope with a dichroic filter set (ex 494nm, em 525nm). Quantification of microvessel density (MVD) was assessed using Roper Scientific Image Pro Plus. 2.16In-vivo imaging of AP mouse after treatment of the nanoparticle 6-week-old C57/BL6 male mice acute pancreatitis model were induced by two i.p. injections of L-arginine (250mg/100 g body weight) as a 8% solution in 0.9% normal saline with an interval of 1 h. After the second injection, the animals were given food and water again. The control mice received an equal volume of 0.9% normal saline (control group, 0h). For verification of the acute pancreatitis model, the injected mice were randomly divided into five groups, and the mice were sacrificed at 12, 24, 48, 72, 96h after the second injection. The pancreases were rapidly dissected for research on H&E stained tissue slice. Blood samples were coagulated and centrifuged at 3000 rpm for 10 min at 4°C, and the serum amylase levels were measured immediately. Free Did, Did labeled CaPi-pDNA-PLGA-NP and Ca-CQ-pDNA-PLGA-NP were administered to C57 mice at 0.2mg kg-1 of Did as i.v. injection through the tail vein. Mice were sacrificed and all major organs incuding heart, liver, spleen, lung, kindey and pancreas were removed and imaged at 4h postinjection by a Quick View 3000 Bio-Real. Only organs that showed a fluorescent signal were used to calculate biodistribution. These include heart, lung, liver, kidney, spleen and pancreas. Percentage of targeting for each organ was calculated as previous report with some modification using pancreas as an example23: %targeting=((fluorescence intensity per unit dry pancreas weight)/sum of fluorescence intensity per unit dry organ weight of all organs)*100%.


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The fluorescence filters were set at ex 655nm and em716nm.

3.Results and discussion 3.1 Preparation and characterization of the Ca-CQ-pDNA complexes Fig. 2 (A) Agarose gel electrophoresis of the Ca-CQ-pDNA complexes with a serious of Ca/CQ ratios; (B) Transfection in vitro in HEK293 with (Ba) the CaPi-pDNA complexes and (Bb) the Ca-CQ-pDNA complexes. Light microscopy phtographs (the left column), RFP fluorescence microscopy photographs (the middle column) and flow cytometry graphs (the right column); (C) Transfection in vitro in CT26 with (Ca) the CaPi-pDNA complexes and (Cb) the Ca-CQ-pDNA complexes, light microscopy photographs (the left column), RFP fluorescence microscopy photographs (the middle column) and flow cytometry graphs (the right column).

3.1.1 Electrophoresis of the Ca-CQ-pDNA complex The molar ratio of calcium chloride to chloroquine (Ca/CQ ratio) was an important parameter of the Ca-CQ-pDNA complex. According to (Fig2 (A)), the influence of the Ca-CQ on pDNA mobility was visually manifested. Slower rate of pDNA migration proved the formation of Ca-CQ-pDNA complex following interaction between Ca-CQ and pDNA. As the Ca/CQ decreased from 2500 to 125, pDNA migrated more and more slowly suggesting greater interaction within the Ca-CQ-pDNA complex. The result suggested that CQ were able to take the place of disodium hydrogen phosphate in the classic calcium phosphate coprecipitation method to condense pDNA. We speculated that the formation of calcium phosphate after ion exchange played the main role in coprecipitaing pDNA and the presence of chloroquine did not interfere with this process. 3.1.2 Transfection in vitro in HEK293 and CT26 with the Ca-CQ-pDNA complexs As the electrophoresis showed, the extent of pDNA condensation in the Ca-CQ-pDNA complex was different when the CQ concentration varied. So, the transfection efficiency of the Ca-CQ-pDNA complexes with a series of CQ concentrations were studied (data not shown), and the highest transfection was obtained with CQ concentration of 2mM. The transfection of 293 cells using the optimal Ca-CQ-pDNA complex formulation was compared with that of the classic calcium phosphate precipitation method (abbreviated as CaPi-pDNA complex) (Fig. 2(B) and 2(C)). As anticipated, the transfection efficiency of the Ca-CQ-pDNA complex was significantly higher than that of the CaPi-pDNA complex, which was due to the enhanced lysosome escape and pDNA protection against degradation caused by chloroquine

24

. In previous study, cells were pretreated with CQ

followed by transfection with calcium phosphate coprecipitation medthods, and exposure to CQ increased the



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transfection efficiency for up to 40% 25. In our study, transfection efficiency of the Ca-CQ-pDNA complex was increased up to 50% in293A cells and 80% in CT26 cells, suggesting the transfection with CQ was more efficient than just pretreatment with CQ. The mechanism of CQ to treat malaria is because the drug can interact with the double helix of DNA by ionic attraction and result in the incapability of DNA replication and RNA transcription 24, 25. As a matter of fact, the increase in transfection of the Ca-CQ-pDNA complex did not conflict with the previous studies. CQ interacts with pDNA through ionic attraction26-29 and the static electrification with the negative phosphate groups on the exterior of the DNA double helix was the first stage before intercalation of CQ into dsDNA double strand helix 28

. In this study, pDNA was mixed with high concentration of calcium chloride solution before mixing with CQ.

The calcium ionic occupied the phosphate group of the pDNA, which prevented contact of CQ with the pDNA. 3.2 The influence of different formulation parameters on physical character of the nanoparticles 3.2.1 The impact on particle size and entrapment efficiency of pDNA Fig.3 Effect of various processing parameters and polymer characteristics on the mean diameter and the entrapment efficiency of the nanoparticles, (A) The initial CaCl2 concentration (M, mol/L); (B) The Ca/CQ ratio in the aqueous phase; (C) The concentration of PVA (%, w/v); (D) The amount of pDNA in the prescription (µg); (E) Sonication power in the primary and secondary emulsifying process (W). All values are reported as mean ±SD (n =3).

The initial concentration of CaCl2 influenced the entrapment efficiency of the pDNA to some extent, while it produced negligible impact on particle size (Fig. 3(A)). When the CaCl2 concentration is low, the poor condensation of pDNA resulted in low entrapment efficiency. As the Ca/CQ ratio increased from 50 to 500, the entrapment efficiency increased gradually (Fig. 3(B)). The high Ca/CQ ratio stands for high phosphate concentration. At Ca/CQ of 50/1, precipitates appeared during the primary emulsifying process, and the precipitate was not able to be encapsulated in the nanoparticle. At Ca/P 250/1 and 500/1, there is no significant difference in both EE and particle size. As the PVA concentration increased, the particle size of the nanoparticles firstly increased and then decreased (Fig. 3(C)). When the PVA concentration is too low (0.5%), the poor emulsifying effect of PVA at this concentration led to relatively large particle size. When the PVA concentration is between 1-3%, the increase of the PVA concentration was helpful for the entrapment efficiency of the pDNA. However, when the PVA concentration further increased to 5%, the elevated viscosity in external aqueous phase resulted in reduced shear


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force which was followed by formation of large drop in the process of emulsification. Along with the increase of pDNA in the prescription, the entrapment efficiency gradually decreased. The pDNA amount was set as 60μg (Fig. 3(D)). The sonication powers for primary emulsification step and secondary emulsification step was investigated, respectively (Fig. 3(E)). The particle size decreased and the entrapment efficiency increased with the increase of the sonication power. Further increase of the sonification power had negative impact on the entrapment efficiency, which was probably due to the degradation of the pDNA under sonification. 3.2.2 The impact of different parameters on pDNA integrity Fig.4 Agarose gel electrophoresis showing the effect of various processing parameters and polymer characteristics on pDNA integrity, (A) The initial CaCl2 concentration (M, mol L-1), (B) The Ca/CQ ratio in the aqueous phase; (C) The amount of pDNA in the prescription (µg); (D) The concentration of PVA (%, w/v); (E) Sonication power in the primary and secondary emulsifying process (W).

Calcium ionic formed well-defined complexes with the phosphate groups of pDNA, which successfully protected the pDNA from degradation during sonication. However, too high concentration of calcium ionic (3.5M) failed to do so probably due to loss of the compact structure under this condition (Fig.4(A)). The result was consistent with the CaPi-pDNA-PLGA-NPs 3. The pDNA band was hardly visible at Ca/CQ 50/1 (Fig. 4(B)). As described previously, when the Ca/CQ was 50/1, precipitates formed during the primary emulsifying process which was not able to protect the pDNA from degradation during sonication. At Ca/CQ of 125/1, 250/1, 500/1, intact pDNA bands were observed suggesting good protection of the pDNA under these conditions. So, Ca/P of 500/1 was adopted for further study. The pDNA remained almost intact under all the sonication powers used in the experiment (Fig. 4(C)). So, the sonication power was selected mainly according to the entrapment efficiency. When the PVA concentration is below 5%, the ideal emulsifying effect had great protection of the pDNA under sonication (Fig. 4(D)). However, when the PVA concentration is 5%, the formation of precipitate resulted in the damage of the pDNA under sonication condition. So, the bands of pDNA were hardly visible. The PVA concentration of 3% was adopted. The sonication power used in this experiment did not significantly influence the pDNA integrity (Fig. 4(E)). The sonication power of 37.5W gave the optimal nanoparticles, which was adopted. Upon the formulation screening, the optimal formulation is as following. 7.5 mg of PLGA 50:50 (MW= 15 kDa)



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was dissolved in 0.75 ml of dichloromethane–ethyl acetate (DCM–EA) (1/1, v/v) as organic phase. The organic phase was emulsified with the mixture of 125ul of calcium precursor solution and 125ul of CQ precursor solution by probe sonication at 37.5W for 50s in ice bath. Then the primary emulsion was added dropwise to 3 ml of 1% (w/v) PVA solution sonicated for 50s followed by rotary evaporation under vacuum to remove the organic solvents at 37°C to get the nanoparticle suspension. The nanoparticle suspension was centrifuged at 4°C with a centrifuge speed of 13,300 rpm for 90 min. The supernatant was discarded and the nanoparticles in the precipitate were dispersed in the ultrapure water for further use. 3.3 XRD, DSC and TEM Fig. 5 XRD patterns of (Aa) CaPi-pDNA complexes, (Ab) Ca-CQ-pDNA-PLGA-NPs, (Ac) plain PLGA-NPs. The DSC curves of (Ba) Ca-CQ-pDNA complexes, (Bb) Ca-CQ-pDNA-PLGA-NPs, (Bc) plain PLGA-NPs. (C ) TEM images of the Ca-CQ-pDNA-PLGA-NPs.

The crystalline nature of the Ca-CQ-pDNA was identified by XRD (Fig. 5(Aa)). The characteristic peaks of Ca-CQ-pDNA was absent in the XRD pattern of Ca-CQ-pDNA-PLGA-NPs (Fig. 5(Ab)) suggesting Ca-CQ-pDNA was in weak crystallization form in the nanoparticles. The DSC thermograms of several samples are shown in (Fig. 5(B)). plain PLGA-NPs have endothermic peaks at 58.6°C and 272.6°C, respectively (Fig. 5(Bc). CaPi-pDNA has an endothermic peak at 190.2°C. Ca-CQ-pDNA complexes had endothermic peak at 39.5°C and 155.5°C (Fig. 5(Ba)). In endothermic figure of Ca-CQ-pDNA-PLGA-NPs(Fig. 5(Bb)), new peaks including 44.8°C, 102.3°C, 186.1°C and 325.8°C arised indicating the interaction between Ca-CQ-pDNA complexes and plain PLGA-NPs. According to TEM, the Ca-CQ-pDNA-PLGA-NPs (Fig.5(C))were spherical in shape. Compared to the plain PLGA nanoparticles (data not shown), the Ca-CQ-pDNA-PLGA-NPs exhibited no significant difference in appearance. 3.4 Toxicity evaluation of the nanoparticles Fig. 6 (A) The cell viability of HEK293 cells treated by the nanoparticles with different concentration in the media (mg ml-1). (B) The hemolysis effect of the Ca-CQ-PLGA-NPs at different concentration (mg ml-1). (C) The H&E staining images of different tissue samples (200X).

The cytotoxicity of the nanopaticles against HEK293 cells was studied (Fig. 6(A)). The cytotoxicity gradually increased as the concentration of the nanoparticles increased from 0.01-8mg-1ml, which was the final


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concentration in the culture media. Usually, the concentration of the nanoparticles in the cell media was below 1 mg-1ml. And the cytotoxicity at these concentrations was negligible. The media with too high concentration of the nanoparticles presented an appearance of emulsion. So, it was reasonable that the cell growth was affected. And the Ca-CQ-PLGA-NPs were more toxic than Ca-PLGA-NPs which was ascribed to the chloroquine embedded inside (p>0.05). The hemolysis rate was under 5% with the nanoparticle concentration from 1.6mg-1ml to 8mg-1ml, suggesting no hemolysis of the nanoparticles(Fig.6(B)). And HE staining of the tissue section suggested that there was no significant toxicity of the nanoparticles against the main organs (Fig.6(C)). 3.5 In vitro plasmid and CQ release Fig. 7 Cumulative release percentage in vitro of (Aa) pDNA and (Ab) CQ from the Ca-CQ-PLGA-NPs over time at different pH conditions. Cellular uptake of (Ba) CaPi-pDNA-PLGA-NPs, (Bb) Ca-CQ-pDNA-NPs at different time points after treatment. The upper panel is the enlarged image of the red circle area in the lower panel. Transfection in vitro in HEK293 cells of (Ca) the CaPi-pDNA-PLGA-NPs and (Cb) the Ca-CQ-pDNA-PLGA-NPs at different time intervals after treatment, light microscopy phtographs (the left column), RFP fluorescence microscopy photographs (the middle column) and flow cytometry graphs (the right column).

The release rate of pDNA and CQ from the nanoparticles was essential for the nanoparticle to exert therapeutic effect in vivo. So, the release behaviors of the pDNA and CQ in media with different pH （pH4.5., 6.8, 7.4） to simulate various physiological conditions were estimated (Fig. 7(A)). The Ca-CQ-PLGA-NPs had no burst release effect of both pDNA and CQ in all of the release media. The pDNA release from the nanoparticles showed typical pH dependent release behavior. At pH4.5, the cumulative pDNA release within 24h was over 30% and eventually reached 60% over 4 weeks. The cumulative release amount at acidic media was 2-4 fold to that at neutral media. The pDNA release at neutral pH was both very low. The cumulative release rate was around 20% (pH7.4) and 30% (pH6.8) at 4 weeks. In conclusion, it was speculated that only very small amount of the pDNA and CQ were released from the nanoparticles before reaching the target site, and quick release would be triggered in acidic condition in endosome after endocytosis. The release of CQ in all conditions was much faster relative to the pDNA. As described above, CQ was able to help pDNA to escape lysosome and prevent pDNA degradation in lysosome. So, it would be definitely helpful to exert its effect if CQ was the first to be released into lysosme,



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3.6 Cellular uptake of the nanoparticles C6-labeled and Did-labeled nanoparticles were both prepared, from which the release of the dyes was below 2% for 24h. So, the C6-labeled and the Did-labeled nanoparticles were used for studying the nanoparticles in vitro and in vivo. Scattered green dots were observed inside of the cells indicating quick endocytosis of the nanoparticles by the cells (Fig. 7(B)). After 2h, in the CaPi-pDNA-PLGA-NPs treated group, the superposition of the fluorescence coming from the NPs and from the lysosome were seen as a yellow colour, suggesting that a part of the nanoparticles entered lysosome of the cells. By contrast, much less yellow colour was seen in the cells treated by the Ca-CQ-pDNA-PLGA-NPs, suggesting that a lot of the nanoparticles had already escaped the lysosome. Similar phenomena were observed at 6h and 12h. Without CQ, the CaPi-pDNA-PLGA-NPs were still entrapped in lysosome till 12h. CQ can induce rapid and pronounced lysosomal vacuole accumulation in cytoplasm22, which was observed in this study. The accumulation of red fluorescence inside of the cells treated by the Ca-CQ-pDNA-PLGA-NPs confirmed this. 3.7 Transfection on HEK293 Previous studies have revealed that the transfection of the gene-loaded PLGA nanoparticles slowly increased from day1 to day7 ascribing to the sustained release rate of pDNA from the PLGA nanoparticles

29, 30

. Similar

phenomenon was observed in this study (Fig. 7(C)). Entrapment of calcium phosphate inside of the PLGA matrix was able to increase the pDNA release rate in the acidic endosome or lysosome. And loading of CQ together with the calcium would futher help the escape of pDNA from lysosome. A significant enhancement in gene transfection efficiency for over 50% was achieved by the Ca-CQ-pDNA-PLGA-NPs relative to the CaPi-pDNA-PLGA-NPs. 3.8 Biodistribution of the nanoparticles in CT26 mouse model Fig. 8 (A) Representative ex vivo ﬂuorescent images of various organs at different points after intravenous injection of the CaPi-pDNA-PLGA-NP (Aa) and the Ca-CQ-pDNA-PLGA-NP (Ab). (B) The total fluorescence intensity of various organs over time, (C) Representative in vivo fluorescent images of mice 4h postinjection treated by 0.9% normal saline, CaPi-pDNA-PLGA-NP and the Ca-CQ-pDNA-PLGA-NP, respectively. (D) The total fluorescence intensity of ROI in tumor area (ROI, Ragion of Interests, red circle in C) of the in vivo image. N = 3 mice per treatment group.

According to the ex-vivo imaging results (Fig. 8(A)), the nanoaprticles were mainly accumulated in the liver due to phagocytosis by reticulo-endothelial system. In the Ca-CQ-pDNA-PLGA-NPs treatment group, total


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fluorescent intensity in tumor was second only to that in liver. And the average fluorescent intensity in tumor was similar to that in liver, which suggests the good tumor targeting of the Ca-CQ-pDNA-PLGA-NPs. In the main organs like liver, the fluorescence was evenly distributed, however, the fluorescence in tumors was very uneven (Fig. 8(Aa)). A lot of research has reported that, though the nanoparitcles were largely accumulated in the tumor region due to the EPR effect, a lot of them were only located in the perivasculature regions of tumors31. It was the high pressure inside of tumors that prevented the penetration of the nanoparticles to the internal part of the tumors31. From 4h to 12h, the fluorescence in some parts of tumors was continuely increased, which suggested the extended circulation time of the nanoparticles in the plasma. By contrast, the average fluorescent intensity in tumor of the CaPi-pDNA-PLGA-NPs treatment group was much less, which was only one half of the Ca-CQ-pDNA-PLGA-NPs (Fig. 8(Ab)). According to the above results in vitro, we hypothesized that inclusion of CQ in the PLGA-based nanoparticles prevented nanoparticle degradation in tumor site through the accelerated lysosomal escape effect. The graph of fluorescence intensity versus time indicates that the fluorescence in the CaPi-pDNA-PLGA-NPs treatment group eliminated faster than that in the Ca-CQ-pDNA-PLGA-NPs treatment group (Fig. 8(B)). The biodistribution of the nanoparticles in CT26 mouse model in vivo at 4h after administration was shown in (Fig. 8(C)), which was consistent with the ex-vivo study. The CaPi-pDNA-PLGA-NPs had obvious accumulation at the tumor site. The fluorescence intensity in tumor site in the Ca-CQ-pDNA-PLGA-NPs treated mouse was around 2-fold relative to that of the CaP-pDNA-PLGA-NPs treated mouse, suggesting greater tumor targeting effect (Fig. 8(D)). This result gives further evidence for the CQ action in vivo. In all, the high accumulation in tumor and sustained clearance of the Ca-CQ-pDNA-PLGA-NP in the experimental animals highlighted the potential for tumor therapy in vivo of the nanoparticle.

3.9 Antitumor activity of ms-Ca-CQ-pDNA-PLGA-NP in vivo Fig. 9 (A) Immunohistochemical analysis of CD31 protein levels in each group including normal saline group(a), vector group(b), the null-Ca-CQ-pDNA-PLGA-NP group, (c) and the ms- Ca-CQ-pDNA-PLGA-NP group (d); (B) CD31 positive MVD levels in frozen sections of the tumor tissues; (C) Representative tumor of each group(n=5); (D) The tumor size of mice bearing CT26 after treatment.

The vector and the null-Ca-CQ-pDNA-PLGA-NP showed anti-tumor effect having significant difference from the normal saline group (p

Facile Construction of Chloroquine Containing PLGA-Based pDNA

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