A Self-Assembled Nanoparticles Platform Based on Pectin

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A Self-Assembled Nanoparticles Platform Based on PectinDihydroartemisinin Conjugates for Co-delivery of Anticancer Drugs Yanxue Liu, Dan Zheng, Yunyun Ma, Juan Dai, Chunxiao Li, Shangzhen Xiao, Kefeng Liu, Jing Liu, Luying Wang, Jiandu Lei, and Jing He ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00842 • Publication Date (Web): 20 Nov 2017 Downloaded from http://pubs.acs.org on November 21, 2017

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ACS Biomaterials Science & Engineering

A Self-Assembled Nanoparticles Platform Based on Pectin-Dihydroartemisinin Conjugates for Codelivery of Anticancer Drugs Yanxue Liu, Dan Zheng, Yunyun Ma, Juan Dai, Chunxiao Li, Shangzhen Xiao, Kefeng Liu, Jing Liu, Luying Wang, Jiandu Lei*, Jing He* Beijing Key Laboratory of Lignocellulosic Chemistry, Beijing Forestry University, Beijing 100083, P. R. China. *Corresponding author, E-mail: [email protected]

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Abstract Natural pectin is an important carrier for delivering drugs in biomedical research, however, there are only a few reports on the preparation of pectin nanoparticles, especially a particle size of below 100 nm with high yield. Here we design pectin-dihydroartemisinin / hydrooxycampothecin nanoparticles (PDC-H NPs) through a self-assembly method. The prepared PDC-H NPs contained hydrophilic part of pectin and hydrophobic anticancer drugs of dihydroartemisinin and hydroxycamptothecin, which could increase drug loading, improve water-solubility, and achieve controlled release of drugs. The results indicated that the particle size of PDC-H NPs was about 70 nm, drug-loaded efficiency of DHA was 20.33 wt% and encapsulation efficiency of HCPT was 14.11 wt%. PDC-H NPs exhibited a higher cytotoxicity, the blood retention time of PDC-H NPs was 4.8-fold longer than DHA and was 6.8-fold longer than HCPT. In addition, effective cellular uptake exhibited an obvious synergistic effect compared with DHA and HCPT. 4T1 tumor-bearing mice also showed a higher survival rate than free DHA and free HCPT. The result show that the self-assembled PDC-H NPs is a promising anticancer drug for Co-delivery. Keywords: Pectin, Nanoparticle, Dihydroartemisinin, Hydroxycamptothecin, Self-assemble, Codelivery


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1. Introduction Chemotherapeutic drugs usually have toxic to human bodies because of slow drug delivery systems, narrow therapeutic index and poor water solubility that easily cleared by the reticuloendothelial system (RES) and mononuclear phagocytic system (MPS)1-3. Commonly, excessive administration of excipients may lead to systemic toxicity such as lipotoxicity, and thus increasing the additional burden for the excretion of non-degradable or harmful carriers. Therefore, drug carriers with biocompatible and biodegradable are urgently designed to solve these drawbacks smartly. Pectin is an important class of natural polysaccharide and is ccomposed of the main chain of galacturonic acid and the neutral sugar side chain, which includes about 65% homogalacturonan (HG), 20-35% rhamnose (RG-I), a small amount of arabinogalactan (AG) and xylogalacturonan (XG)4-7. The excellent bioavailability and bioactivity of pectin can improve the body immunity, modulate immunological responses, inhibit tumor growth and metastasis and induce apoptosis8, 9. The antitumor activity of pectin is mainly due to the recognition and intervention of galactolectin-3 (Gal-3) ligands. A large amount of terminal galactose residues is recognized and easily expressed on hepatic cell surfaces for achieving liver-targeted

8, 10, 11

. In addition, the modified

pectin can enhance the ability of biological adhesion with biological tissues to improve bioavailability of hydrophobicdrugs1, 12-14. Recently, pectin was modified to prepare pro-drug15, hydrogel16, microgel17. For example, pectin-4-aminothiophenol (Pec-ATP) microparticles were developed for specific killing Caco-2 cells, the obtained particle size was 5.16 ± 2.41 µm and the corresponding drug loading rate was 1.15 ± 0.03%. Blood circulation time of Pec-ATP microparticles was 34.4-fold longer than free metronidazole18. Moreover, pectin–ketoprofen (PT-


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KP) prodrug was proposed for targeting colon cancer with lower plasma concentrations and higher transmission capacity19. Hu et al. developed a core–shell biopolymer of zein–pectin nanoparticles for encapsulating curcumin with a particle size of 250 nm. However, the considerable size, uncontrollable drug release and low yield were all not conducive to the transmission and clinical application of pectin-based drugs20. Our groups have reported many drug delivery systems used to carrier hydrophobic drugs 2123

, however, little work has been reported to prepare satisfactory pectin-based nanoparticles for

hydrophobic drugs delivery, especially containing synergistic effect of two anticancer drugs for co-delivery. The existing drug delivery systems have many disadvantages that limit the application of pectin. Accordingly, more innovative approaches are urgent proposed to broaden the functions and applications by coupling small molecule drugs to pectin24-27. Desirable EPR effect, high drug loading capacity, controlled drug release, biodegradable, either eliminate or minimize toxicity of the carrier are also used for evaluating pectin drug delivery system28. In this work, we thus design a pectin-based nanocarrier as a combination strategy for simultaneously

multiple-cargo

(hydrophobic

drugs

dihydroartemisinin

and

10-

hydroxycamptothecin) delivery to tumor sites. First, pectin is directly coupling with dihydroartemisinin (DHA) to form pectin-dihydroartemisinin (PDC) pro-drug using 1-ethyl-3-(3dimethylaminopropyl)-carbodiimidehydrochloride (EDC) as an activator in pyridine. Second, achieving the goal of combination therapy and co-delivery, another hydrophobic anticancer drugs 10-hydroxycamptothecin (HCPT) was chosen for entrapping into the PDC to form PDC-H NPs, and synergistic treatment of cancer by self-assembly. Drugs directly connected to the pectin could increase drug loading capacity because of rich carboxyl groups in pectin23. The prepared hydrophobic core containing two kinds of anticancer drugs with an enhanced hydrophilic surface


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layer of pectin to stable the nanoparticle in neutral aqueous solution environment. The obtained nanoparticles were characterized by assessing particle size, drug loading and embedding rates. Furthermore, antitumor activities of PDC-H NPs were also investigated in vitro and in vivo.


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2. Materials and Methods 2.1 Materials Low-methoxy pectin from citrus peel (DE= 36%) was purchased from USA, and is FDA and EU foodgrade materials. Dihydroartemisinin (DHA) and 10-hydroxycamptothecin (HCPT) were purchased from Chengdu Preferred Biotechnology Co.,Ltd (Chengdu, Sichuan, China). Dimethyl sulfoxide, acetonitrile, diethyl ether, ethanol, pyridine, glacial acetic acid, 4dimethylamino pyridine (DMAP) ， 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide HCl (EDC) and were purchased from Sigma Aldrich (Oakville, ON, CA). Fetal bovine serum was purchased from HyClone. Penicillin and streptomycin, Gibco Dulbecco’s Phosphate-Buffered Saline (DPBS) and Roswell Park Memorial Institute (RPMI-1640) were bought from Invitrogen. Cell-Counting Kit-8 was supplied by Dojindo Laboratories. MCF-7 as a widely studied human epithelial cancer cell line derived from breast adenocarcinoma and the mouse breast cancer cell line (4T1) were obtained from 410.4 tumor strain without mutagenesis screen 6-thioguanlinol resistance cell line, purchased from the Peking University Health Science Center (Beijing, China), were maintained in DMEM and added in 10% fetal bovine serum at 37 °C and 5% CO2 for 10 passages. Female BALB/c mice, 4-6 weeks of age, were purchased from Beijing Hfk Biosciece Co., Ltd. The animal experiments were consistent with the guidelines of the National Institutes of Health (NIH Publication No. 85-23, revised 1985), and the Experimental Animal Ethics Committee permission, Beijing.


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2.2 Synthesis of pectin-dihydroartemisinin conjugate (PDC)

Figure 1. Synthesis of the pectin PDC. Note that the distribution of DHA substitutions is random. Pectin-dihydroartemisinin conjugate was synthesized by the scheme of Figure 1. DHA (500 mg, 1.76 mmol), pectin (500mg, 3.33 mmol), DMAP (50 mg, 0.41 mmol), EDC (230 mg, 1.20 mmol) were dissolved in pyridine and passed through nitrogen at 35 °C for 48 h. The reactants were precipitated in acetonitrile (1:3, v/v) with a centrifugal rate of 4000 rpm for 30 min, precipitation was collected by repeatedly washed with acetonitrile. The resulting precipitate was dialyzed (MWCO 2 KDa) in deionized water for 24 h and changed the dialysis liquid three times interval of 4 h. Dialysis substance was freeze-drying to obtain PDC powder. Pectin was dissolved in deuterium water (D2O), DHA and PDC were dissolved in deuterated chloroform (CDCl3), and were analyzed on a Bruker 500 instrument.


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2.3 Preparation of pectin nanoparticles (PDC-H NPs) PDC-H NPs was prepared by self-assembly method as described previously11, 29, 30. In order to

achieve

a

double-drug

delivery

system,

another

hydrophobic

anticancer

drugs

hydroxycamptothecin was chosen for entrapping into the PDC NPs by self-assemble. Simply, 10 mg PDC and 2 mg HCPT were mixed in 0.5 mL dimethyl sulfoxide, and dropped slowly into a rotating solution of 10 mL PBS solution (pH 7.4) for 15 min to prepare PDC-H NPs. The obtained nanoparticles solution was dialyzed (MWCO: 3500 KDa) against 200 mL PBS solution, and exchanged the dialysis solution every 6 h with the same volume of PBS solution for four times. Then the solution of nanoparticles was freeze-dried to get powder. The preparation method of PDC NPs is the same as PDC-H NPs. The particles size was determined by dynamic light scattering using a particle analyzer (Zetasizer Nano-ZS, Malvern Instruments Ltd, Malvern, UK). Each measurement was repeated three times, and the results were processed with DTS software version 3.32. 2.4 Determination of drug loading efficiency and encapsulation efficiency The content of DHA in PDC NPs was test by UV-Vis spectrophotometer.31 DHA was dissolved in 75 mL,60 wt % ethanol-water solution and filtered. 5ml filtrate was mixed with 23 mL, 2 %wt of NaOH solution, and kept in a 60 °C warm water bath for 30 min. DHA was determined at 238 nm for five different concentrations ranging from 10 to 200 µg mL-1 by UV absorbance. The pretreatment of PDC was consistent with pure DHA. The drug concentration of DHA and HCPT were calculated by the standard curve: Y1=0.00737XDHA, R2=0.9998; Y2=0.03398XHCPT, R2=0.9998. Where Y1 and Y2 represent the absorbance of the sample, X represents the concentration of the sample,µg / mL.


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In PDC-H NPs, contents of DHA and HCPT were also assayed by UV-Vis spectrophotometer at 238 nm 254 nm, respectively. Drug loading efficiency (DLE) of DHA and encapsulation efficiency (EE) of HCPT in PDC-H NPs were calculated according to the following equation: DLE (%) = (weight of the drug in nanoparticles / weight of nanoparticles) ×100%

(1)

EE (%) = (Weight of HCPT in nanoparticles / Initial amount of drug) ×100%

(2)

2.5 Determination of drugs release in vitro The drug release of DHA and HCPT in PDC-H NPs were analyzed by a dialysis method. 10 mL of 5 mg mL-1 PDC-H NPs solutions was diluted adjusted to pH 5.0, 7.4, 8.0, placed in dialysis bags (MWCO 3.5 kDa) to immerse a 200 mL PBS of pH 5.0, 7.4, 8.0 at 37 °C with slightly oscillation. 2 mL PBS medium was taken out at timed intervals, and was determined by the HPLC method (HCPT: 254 nm, 30: 70 mixtures (v/v) of acetonitrile-water as a mobile phase, flow rate of 0.8 mL min-1; DHA: 238 nm, 45: 55 mixtures (v/v) of acetonitrile-water, flow rate: 0.8 mL min-1) through a C18 reverse phase column. 2.6 Hemolysis assay There are 98% identical genes between rats and human. Furthermore, rats’ lines are pure and easily compare that can eliminate the interference factors of different germs and reduce the incidence of spontaneous tumors. In addition, changes in blood pressure and vascular resistance in rats are sensitive to drug effects. Therefore, rats have been widely used in hemolysis assay29, 30

. Briefly, 10 mL fresh blood samples from healthy rats were immediately mixed with EDTA-

Na2, and collected red blood cells by centrifuging. The obtained red blood cells were diluted into


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a concentration of 5 × 108 cells mL-1 with ice-cold DPBS. Nanoparticles solution were diluted into a concentration of 0.1 mg mL-1 to 1 mg mL-1 and assimilated, respectively, and 1 mL erythrocyte suspension was added to incubate at 37 °C for 1 h. The precipitate was removed by centrifugation at 1500 rpm at 4 °C for 15 min. The release of hemoglobin in supernatant was determined at 541 nm by using an infinite M200 microplate spectrophotometer (Tecan, Switzerland). Then, DPBS was used as a negative control (0% lysis) and 1% Triton X-100 in DPBS was used as a positive control (100% lysis). Hemoglobin release was calculated as: (ODsample - ODnegative control) / (ODpositive control - ODnegative control) × 100%

(3)

Hemolysis of nanoparticles was measured though three independent experiments. 2.7 Toxicity analysis in vitro Before doing toxicology test, 4T1 cells and MCF-7 cells were synchronized as reported earlier32,

33

. Briefly, 4T1 cells and MCF-7 cells were grown in DMEM and RPMI-1640,

respectively, and 5% FBS and antibiotics were added in the well plate at 37 °C. 4T1 cells and MCF-7 cells were density-arrested by plating at 5 × 105 cells/cm2 in FBS for 48h and trypsinized, which was repeated at low density of 1 × 105 cells/cm2. Afterwards, cells were assayed through propidium iodide Fluorescence activated Cell Sorting (FACS). CCK-8 assay was used for cell viability evaluation of different samples26, 34. 4T1 cells were diluted into a concentration of 4 × 103 cells / well, seeded in a 96-well plate (Corning, USA) and incubated 24 h. After that, the prepared DHA, HCPT, PDC NPs and PDC-H NPs were added in cells at 37 °C for 72 h. The amount of DHA were ranged from 0.8 to 100 µg mL-1, and nanoparticles were the same as DHA. HCPT dose was equal to the amount of embedding in PDC-H NPs. 20 µL CCK-8 solution was added to the well plate and incubated at 37 °C for 2h. Detection of cell viability at


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450 nm though an infinite M200 microplate spectrophotometer. IC50 was calculated as reported earlier by the Boltzmann sigmoidal function from Origins 8.6 (OriginLab, Northampton, USA)35. The synergistic effects of DHA and HCPT in the PDC-H NPs was evaluated by the combination index (CI): CI = DHA1 / DHA0 + HCPT1/HCPT0

(4)

Whereby DHA1 and HCPT1 represent the IC50 of DHA and HCPT of PDC-H NPs, and DHA0 and HCPT0 represent IC50 of DHA and HCPT. CI 1 shows an antagonistic effect. 2.8 Cellular uptake Cells uptake of free HCPT and the embedding HCPT were detected by the absorbance of 488 nm on confocal laser scanning microscopy (CLSM, TCS SP5, Leica). Simply, 4T1 cells were trypsinized for 1 min, added the culture medium, dispersed evenly, and centrifuged to obtain cell pellets. Cells were repeatedly suspended at a density of 1.0 × 105 cell/mL, and taken 1 mL into 4 cm2 confocal petri dishes and incubated overnight at 37 °C. A certain concentration of HCPT (IC50) and PDC-H NPs (IC50) was added and incubated for 4 h. After that, excessive liquid in cells was removed and washed for many times. The cleaned cells were fixed with 4% formaldehyde solution for 15 min, and added 1 mL DAPI solution with the concentration of 0.5 µg/mL for 5 min. The obtained cells were washed repeatedly with DPBS, centrifuged to remove supernatant, finally dispersed in DPBS solution at a certain concentration and kept away from light at 4 °C. 2.9 Pharmacokinetics for mouse


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24 tumor-free healthy mice were randomly allocation and injected drugs via the tail vein. Blood samples were collected at 0, 0.1, 0.25, 0.5, 1, 2, 4, 8, 12, 16, 20, 24, 36 and 50 h from the orbital plexus, and centrifuged immediately to obtain plasma at 4 °C for 10 min. 50 µL of 0.1 N NaOH was added into 100 µL plasma with a constant temperature of 37 °C for 15 min to determine the total DHA or HCPT, and added into 50 µL of 0.1 N HCl, 100 µL methanol, obtained supernatant by centrifugation at 4000 rpm for 5 min. 100 µL of methanol was mixed with supernatant and measured by HPLC (C18, 5 mm, 4.6 × 250 mm)

36

. The blood levels of

DHA or HCPT was painted through the unit of percentage of injected dose per gram (% ID/g) against time after injection. 2.10 Subcutaneous tumor efficacy models Evaluation of in vivo animal efficacy was implemented through characterizing tumor volume (TV), the relative tumor volume (RTV) and Tumor Growth Inhibition value (TGI). 30 BALB/c female mice (6-7 weeks) were injected 1 × 106 4T1 cells/mouse in the right auxiliary flank region. When tumors volume was about 50 mm3, mice were administered intravenous injection with PBS (control), free DHA (5 mg kg-1), free HCPT (2 mg kg-1), PDC NPs and PDCH NPs were equal to DHA concentration on days 0, 2, 4, 6, and 8, respectively. The tumor sizes and body weights of mice were monitored before each administration. The tumor volume was calculated using the following formula: Tumor volume (TV) = (L × W2)/2

(5)

RTV was calculated for different time points before each administration, and %TGI was calculated using the following formula:


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%TGI = [1-(RTVdrugs / RTVcontrol)] ×100%

(6)

Where RTVdrugs is the relative tumor volume of the treatment group, RTVcontrol is the relative tumor volume of the control group. 2.11 Detection of allergic reaction Evaluation of allergic reaction is vital for prevention of human allergies before administration. 30 tumor bearing mice were randomly divided into five groups (control, DHA, HCPT, PDC NPs and PDC-H NPs) and administered via tail intravenous injection every two days (DHA: 5 mg kg-1; HCPT: 2 mg kg-1; PDC NPs, and PDC-H NPs were equal to DHA concentration). After treatment, blood from five groups of mice was collected and centrifuged to obtained serum samples which were analyzed by the procedure of Mouse IgE ELISA. 2.12 Statistical analysis The statistical analysis was performed by analysis of variance (ANOVA). All graphical data is reported as mean ± standard deviation (SD). Significance levels were set at * p < 0.05.


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3. Results and discussion 3.1 Preparation of pectin-dihydroartemisinin conjugate In this article, PDC was synthesized following the scheme route reported in Figure 1. a hydrophilic pectin and hydrophobic drug DHA were synthesized via ester bonds. Figure 2 shows the 1H-NMR spectra of DHA (CDCl3), pectin (D2O) and PDC (D2O), where the signals at δ 3.22–4.01 are attributed to the most characteristic peak protons of pectin, and the signals at δ 0.80–2.90 are attributed to the characteristic peak protons of DHA. The characteristic peak of DHA (δ 0.95 ~ 2.12) was clearly seen in the 1H-NMR spectra of PDC. In addition, methyl proton peak from δ 0.96 moved to δ 1.10, and the methine proton peak δ 5.12 was found in PDC. However, the formation of ester bonds between pectin and DHA wasn’t very obvious because of the weakened signals between DHA and pectin in D2O. 3.2 Drug loading and encapsulation efficiency Hydrophobic drug HCPT is sensitive to pH and displayed significantly broad-spectrum antitumor activity36-38. As shown in Figure 3, the UV absorbance spectra of DHA was 238nm, the same absorbance spectra were presented in PDC, and absorption tail peak of pectin could be also observed. As showed in Table 1, the drug-loaded rate of PDC-H NPs was 16.19% (DHA), and encapsulation efficiency of 13.05% (HCPT). The obtained PDC-H nanoparticle size was about 71 nm according to the optimal drug loading rate, which was a better choice for DHA wt% feeds. The larger nanoparticles are easily cleared by the reticuloendothelial system, and the smaller nano-objects prolong the retention time in the bloodstream. In particular, nanoparticles diameter below 100 nm have been considered to be an ideal excipient for cancer treatment due to


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their favorable bio-distribution, clearance and accumulation capacity39-41.

Figure 2. 1H-NMR spectra of pectin and PDC in D2O, DHA in CDCl3. Table 1 Particle size, drug loading efficiency and Zeta potential of nanoparticles Compound

DLEDHA (wt%)

DLEHCPT (wt%)

Size (nm)

Zeta potential (mV)

PDC NPs

19.17 ± 1.23

—

59 ± 8.19

6.51 ± 0.22

PDC-H NPs

16.19 ± 1.16

13.05 ± 1.07

70 ± 9.31

7.56 ± 0.25


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Figure 3. Analysis of DHA content in PDC by UV method. 3.3 Stability of PDC-H NPs The principle of polymer deposition method by self-assembly were shown in Figure 4. Simply, HCPT was chosen as a fluorescence anticancer drugs of water-insoluble to be encapsulated into the PDC, and formed PDC-H NPs by self-assembly, containing the hydrophilic shells and hydrophobic nuclei. When free HCPT was loaded to form PDC-H NPs, the increased particle sizes and the enhanced hydrophobic effect of core were due to the insertion of the hydrophobic drug into the nanoparticles (Table 1, Figure 5). Seen from Figure 5a and 5b the PDC-H NPs had a good dispersibility, and the distribution of particle sizes is relatively concentrated (40-80 nm), which implied a relatively satisfactory results for drug delivery. In addition, the particle size for PDC NPs and PDC-H NPs was observed in an interval of 1- 2 days. Seen from Figure 6, PDC-H NPs presented a smoother trend and smaller change than PDC NPs in PBS which caused by the hydrophobic effect of HCPT.


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The zeta potential is applied for evaluating the stability and dispersion of nanoparticles. Most charged functional groups can interact with the active nanoparticles of cells. Positively charged particles have a great efficiency in cell membrane infiltration and intracellularization, and are the main platform for drug delivery. Obviously, PDC-H NPs all have a positive surface charge (ζ = 7.56 mV) in Table 1 because low ester pectin is rich in galacturonic acid and have weak acidity. When the polymer is self-assembled to form nanoparticles, a small amount of unreacted galacturonic acid is exposed to the outside to form a positive charge. As we expected, PDC-H NPs was more stable than PDC NPs.

Figure 4. Schematic design of PDC-H NPs. PDC was synthesized by directly introducing drug molecules DHA into pectin, and then self-assembled into PDC-H NPs with free HCPT being encapsulated.


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Figure 5. (a) TEM images and (b) the particle size distribution of PDC-H NPs. Scale bars were 100 nm.

Figure 6. The stability of nanoparticles for 26 days of storage at 4 °C (*p HCPT > PDC-H NPs (Table 2). The cytotoxicity of PDC-H NPs was 88.4-fold greater than free DHA in 4T1cells, and was 168.5-fold greater than free DHA in MCF-7 cells. Otherwise, the cytotoxicity of PDC-H NPs was 18.8-fold greater than free HCPT in 4T1cells, and was 10.8-fold greater than free HCPT in MCF-7 cells. It’s clearly revealed that PDC-H NPs was more effective than free DHA and HCPT in vitro toxicity test. Moreover, increased nanoparticles’ toxicity was different between 4T1 cells and MCF-7 cells due to chemosensitivity of different cell lines and drugs’ slowly release. In addition, polymers internalization and strongly release in lysosomes may further enhance drug’s efficacy. In the PDC-H NPs (15.19 wt% DHA and 13.05 wt% HCPT) IC50 of 4T1 and MCF-7 were 0.17 µg mL-1 and 0.44 µg mL-1, and the calculated combination indices (CIs) of DHA and HCPT in the PDC-H NPs was 0.05, which suggested that PDC-H NPs


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had achieved a considerable synergistic effect by co-delivery of DHA and HCPT. Furthermore, the results also revealed that the nanoparticles had high cytotoxicity than free drugs. Table 2 In vitro cytotoxicity analysis (IC50, µg mL-1). Note: PDC-H NPs in 4T1 cells and MCF-1 cells have significant differences comparing to DHA. Compound

4T1 cells

MCF-1 cells

DHA

6.19 (0.5109)

7.35 (0.7725)

HCPT

0.33 (0.0529)

0.68 (0.0451)

PDC NPs

2.16 (0.2140)

2.44(0.2913)

PDC-H NPs

0.07 (0.0133)*

0.12 (0.0283)*


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Figure 9. Cellular cytotoxicity of DHA, HCPT, PDC NPs and PDC-H NPs in 4T1 cells and MCF-7 cells. Cell viability of 4T1 cells (a) and MCF-7 cells (c) was measured by CCK-8 assay, DHA (4 mg/mL), HCPT (5 mg/mL), and PDC NPs and PDC-H NPs (equivalent to native DHA); DHA, HCPT and nanoparticles were diluted to different concentrations and administrated in 4T1 cells (b) and MCF-7 cells (d) (n = 3, error bars represent standard deviation). Note: Cell viability of 4T1 cells (a) and MCF-7 cells (c) treated with HCPT and nanoparticles (equivalent to native DHA) are significant differences comparing to DHA. 3.7 Cellular uptake Nanotechnology-based drug delivery systems have shown significant promise in breast cancer cells therapy45. Otherwise, nanoparticle delivery of multiple drugs has indicated that synergistic effects in the combined treatment of cancer. 4T1 cells were incubated with free HCPT (IC50) and PDC-H NPs (IC50) for 4 h to evaluated the cellular uptake efficiencies by CLSM. As shown in Figure 10 a, b, the green fluorescence of HCPT and PDC-H NPs and the blue fluorescence of DAPI were visualized under fluorescence confocal microscopy. PDC-H NPs increased green fluorescence was more uniformly and densely located around the cytoplasm attached to the surface of cancer cells than free HCPT, indicating a high delivery efficiency in 4T1 cells through visualization.


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Figure 10. Confocal microscopic of 4T1 cells treated with (a) PDC-H NPs and (b) free HCPT with free HCPT (IC50) and PDC-H NPs (IC50) at 37 °C for 4 h. 3.8 Pharmacokinetics experiment The pharmacokinetics study was undertaken by intravenous administrations of DHA, HCPT, PDC NPs and PDC-H NPs injection to 4T1-bearing mice. As is shown in Figure 11, DHA concentrations in plasma gradually declined with the passage of time after injecting preparation by intravenous administrations (Figure 11a). HCPT concentrations in PDC-H NPs was similar to DHA-loaded nanoparticles (Figure 11b). Obviously, the blood circulation time of DHA in PDC-H NPs (55 h) was maintained a higher concentration in plasma than free DHA (12 h), and the embedded HCPT in PDC-H NPs (30 h) was also higher than free HCPT (8 h). However, in the blood circulation process, the concentrations of free DHA and free HCPT in plasma rapidly disappeared after intravenous injection and below 5%. PDC-H NPs still exhibited a prolonged clearance ability with the DHA levels of 15.44% ID per g at 24 h after administration. PDC-H NPs could prolong the blood circulation half-time of DHA from 1.0 h to


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4.8 h, which was 4.8-fold longer than free DHA. PDC-H NPs could prolong the blood circulation half-time of HCPT from 0.4 h to 2.7 h.

Figure 11. Blood circulation curves and half-time of drugs. PDC-H NPs compared to free DHA (a), and PDC-H NPs compared to free HCPT (b). Error bars were calculated by six mice per group at each time point. 3.9 In vivo antitumor activity studies of PDC-H NPs In xenograft models of bread tumor, in vivo antitumor efficiency of nanoparticles compared with DHA and HCPT was at equivalent doses of 8 mg kg-1 DHA and 6 mg kg-1 HCPT, respectively (Figure 12a). After 40 days’ treatment, a significant difference was made in tumor volumes of the mice between free drugs groups and nanoparticles preparation groups in Figure 12b. Anti-tumor capacity of DHA, HCPT, PDC NPs and nanoparticles was PDC-H NPs > PDC NPs > DHA and free HCPT. Survival rate of PDC-H NPs were 80.2% (24 days) and 89.5% (20 days), while DHA was 11.5% (24 days) and 33.9% (20 days), and HCPT was 9.6% (24 days) and 28.1% (20 days) (Figure 12c, Table 3). Results suggested that the tumor volumes of the PDC-H NPs treated group were extremely smaller than those treated with DHA and HCPT injection. These findings were coincided with the results of evaluations in vitro. The mice’s average body weights had no obvious changes in all treated mice, which suggested the


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nanoparticle as a drug was safe at such a dose(Figure 13a). Although the increased tumor volume has an effect on the weight of mice, the effect on body weight is not significant due to the tumor volume of mice is much smaller than that of mice. Therefore, the prepared PDC-H NPs is significantly inhibition of the tumor with small changes in body weight46, 47.

Figure 12. In vivo antitumor activity of free DHA, free HCPT, and nanoparticles in the subcutaneous mouse model of 4T1 cells. (a) Tumor photographs of each treatment group on day 24. (b) Relative tumor volumes of different groups after administration; (c) Survival rate of different groups after administration.


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Figure 13. (a) Mice weights were recorded over the 24-day observation; (b) IgE levels of mice treated with free DHA, free HCPT, and nanoparticles for 30 min; (c) White blood cell (WBC) changes after administrations with free DHA, free HCPT, and nanoparticles. The blood sample was collected from mice till the last dosage treatment. Data as means ± SD; n = 6. Note: IgE levels of mice treated with nanoparticles are significant differences comparing to the free DHA and free HCPT; WBC changes of nanoparticles are significant differences comparing to free DHA and free HCPT. Table 3 4T1xenograft model (q2d ´ 5): efficacy comparison.

a

indicate Mean tumor volume

(TV), RTV, and %TGI date were taken at day 20; b indicate % Cures were taken at day 24. Compound

Mean TV ± SDa (mm3)

RTVa

TGIa (%)

Curesb (%)

Control

5134 ± 2014

43.0 ± 18.7

0

0

DHA

3057 ± 1322

27.9 ± 13.1

33.9

11.5

HCPT

3382 ± 1586

26.8 ± 11.5

30.1

9.6

PDC NPs

814 ± 223

8.3 ± 3.2

82.4

60.9

PDC-H NPs

551 ± 195

4.9 ± 1.9

89.5

80.3

3.10 Evaluation of the side effects


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Although DHA prodrugs were reported to have significant therapeutic effects in vivo, nonnegligible adverse effect was still an urgent problem to be resolved. Type I hypersensitivity is the most common type of the hypersensitivity reaction, which has the significant individual differences and genetic predisposition that means rapidly reaction and fast regression. In this paper, parameter lgE levels of DHA, HCPT, PDC NPs and PDC-H NPs were selected for rapid evaluation of hypersensitivity reactions. The blood IgE levels of mice in different groups (DHA, HCPT, PDC NPs, and PDC-H NPs) are shown in Figure 13b. Administration with free DHA and free HCPT had a higher lgE level than the control group and PDC NPs and PDC-H NPs groups. Therefore, nanoparticles showed a great advantage in reducing the risk of hypersensitivity reactions substantially. The blood toxicity with different formulations treatment was measured by testing the WBC count. From Figure 13c, the total WBC count of mice treated with free DHA showed a little decrease over the normal groupexcept nanoparticle formulations. It’s obviously clarified

that

PDC

NPs

and

PDC-H

NPs

could

avoid

severe

hematotoxicity.


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4. Conclusion Our study highly suggests that the combined treatment can achieve synergistic anticancer effects, and may be an attractive strategy for breast cancer. The self-assembled PDC-H NPs with a smaller particle size of ~70 nm (DHA: 20.33 wt%, HCPT: 14.11 wt%). In vitro assays demonstrated that the PDC-H NPs exhibited more cellular uptake, stronger cell apoptosisinduction and cell-viability inhibition ability than DHA and HCPT. PDC-H NPs could actively capture 4T1 cells by confocal microscopy imaging. The nanoparticles technology of this paper reminded is also suitable for other hydrophobic drugs, and the development of other functional groups on this conjugate remains to be urgently resolved.


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Acknowledegments This study was supported by the National Natural Science Foundation of China (No. 21576029; 21406013).


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For Table of Contents Use Only Title: A Self-Assembled Nanoparticles Platform Based on Pectin-Dihydroartemisinin Conjugates for Co-delivery of Anticancer Drugs Authors: Yanxue Liu, Dan Zheng, Yunyun Ma, Juan Dai, Chunxiao Li, Shangzhen Xiao, Kefeng Liu, Jing Liu, Luying Wang, Jiandu Lei*, Jing He*


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Figure 1 146x130mm (300 x 300 DPI)


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Figure 2 146x131mm (300 x 300 DPI)



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Figure 3 104x78mm (300 x 300 DPI)


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A Self-Assembled Nanoparticles Platform Based on Pectin

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