Enhancing Doxorubicin Delivery toward Tumor by ... - ACS Publications

Subscriber access provided by UB + Fachbibliothek Chemie | (FU-Bibliothekssystem)

Article

Enhancing doxorubicin delivery towards tumor by hydroxyethyl starch-g-polylactide partner nanocarriers Chan Yu, Qing Zhou, Fan Xiao, Yihui Li, Hang Hu, Ying Wan, Zifu Li, and Xiangliang Yang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b00048 • Publication Date (Web): 07 Mar 2017 Downloaded from http://pubs.acs.org on March 12, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Enhancing Doxorubicin Delivery Towards Tumor by Hydroxyethyl Starch-g-Polylactide Partner Nanocarriers Chan Yu1,, Qing Zhou1,, Fan Xiao1, Yihui Li1, Hang Hu1, Ying Wan1*, Zifu Li1,2, Xiangliang Yang1,* 1

National Engineering Research Center for Nanomedicine, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, P. R. China 2 Wuhan Institute of Biotechnology, High Tech Road 666, East Lake High Tech Zone, Wuhan, 430040, P. R. China

These authors contributed equally to this work.

*Corresponding authors: Dr. Ying Wan Tel + 86 27 87792147 Fax + 86 27 87792234 E-mail: [email protected] (Y. Wan)

Dr. Xiangliang Yang Tel + 86 27 87793539 Fax + 86 27 87792234 E-mail: yangxl@ hust.edu.cn (X. Yang)

Key words: hydroxyethyl starch copolymer, polylactide, nanoparticle, doxorubicin, partner carrier, cancer chemotherapy

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 44

Abstract: Doxorubicin (DOX), a kind of wide-spectrum chemotherapeutic drug, can cause severe side effects in clinical use. To enhance its antitumor efficacy while reducing the side effects, two kinds of nanoparticles with desirable compositions and properties were assembled using optimally synthesized hydroxyethyl starch-grafted-polylactide (HES-g-PLA) copolymers and utilized as partner nanocarriers. The large empty HES-g-PLA nanoparticles (mean size: ca.700 nm), at an optimized dose of 400mg/kg, were used to block up the reticuloendothelial system in tumor-bearing mice 1.5 h in advance, and the small DOX-loaded HES-g-PLA nanoparticles (mean size: ca.130 nm) were subsequently applied to the mice. When these partner nanocarriers were administered in this sequential mode, the released DOX had significantly prolonged plasma half-life time, much slower clearance rate as well as largely enhanced intratumoral accumulation as compared to free DOX. In vivo antitumor studies demonstrated that the DOX-loaded HES-g-PLA nanoparticles working together with their partner exhibited remarkably enhanced antitumor efficacy in comparison to free DOX. In addition, these HES-g-PLA partner nanocarriers showed negligible damage to the normal organs of the treated mice. Considering safe and efficient antitumor performance of DOX-loaded HES-g-PLA nanoparticles, the newly developed partner nanocarriers in combination with their administration mode have promising potential in clinical cancer chemotherapy. 1. INTRODUCTION Chemotherapy is one of important strategies for treating different cancers.1 Although a variety of chemotherapeutic drugs have been developed for cancer treatments, the efficacy of these drugs is often limited mainly due to lack of specificity and short half-life time.1,2 In addition, most currently available chemotherapeutic drugs can cause various side effects.1-3 Among commonly used chemotherapeutic drugs, doxorubicin (DOX) has been regarded as an effective one.4 DOX is an anthracycline glycoside antibiotic with wide-spectrum antitumor 1


Page 3 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


activity against a range of malignancies, including tumors occurred in breast, endometria, liver, bile duct and esophagus.5-8 Nevertheless, the use of DOX is often limited owing to its low specificity towards tumor and severe side effects. In particular, DOX-induced myocardial impairment could potentially lead to heart failure.4-8 Accordingly, there is a pressing need to develop suitable carriers for delivering DOX specifically towards tumor while alleviating its side effects. Liposomes are often used as vehicles for delivering chemotherapeutic drugs.9,10 Nevertheless, liposomes have some intrinsic shortcomings, such as low drug load efficiency and premature drug leakage, and more importantly, they can be easily eliminated by the reticuloendothelial system (RES) without fulfillment of their missions.9,10 Therefore, polyethylene glycol (PEG) modified liposomes have been developed to minimize the opsonization and clearance of liposomes during their blood circulation.9,10 One of them, the PEGylated liposome loaded with DOX, known as DOXIL®, has already been used in clinic.11,12 However, it is found that DOXIL®-involved treatments also induce side effects such as hand-foot syndrome and infusion-related responses.13-15 An effort is thus made to load DOX into another type of PEGylated polymeric nanoparticles (NPs), also referred as NK911. NK911 exhibits its anticancer efficiency similar to that of DOXIL®, and commendably, the mentioned side effects caused by DOXIL® have not been observed in the clinical trials of NK911,16 implying that polymeric NPs could serve as a better carrier for the DOX delivery as compared to liposomes. PEGylation technique is commonly used to modify NPs for prolonging their circulation time and control their dosing interval.17-19 However, this technique has raised several concerns. PEG is a non-degradable polymer and it can cause unfavorable effects when used at high parenteral doses or for a long period of time.20 It has been reported that high intracellular PEG accumulation can alter organelle density, and concomitantly, give rise to variations in the 2



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 44

activity of lysosomal enzymes and transporters as well as membrane glycoproteins.20,21 Besides these, increasing stability of NPs arisen from PEGylation, to a large extent, could impede the escapement of drugs from endosome, and the internalized drugs in tumor cells would not be able to function efficiently.17,20,21 In an attempt to circumvent “PEG dilemma”, several natural polysaccharides, including heparin, dextran and hydroxyethyl starch (HES), have been explored as substitutes for modifying NPs.22-24 Among them, much attention has been paid to HES in recent years.25-28 HES is a biocompatible and biodegradable polysaccharide and has been used as a plasma volume expander.29 Growing evidence suggests that HESylated nanocarriers have ability to prolong the circulation time of loaded drugs with enhanced efficiency, and HESylation technique is a possible replacement for PEGylation without incurring the disadvantageous effects caused by PEG because of fully biodegradable nature of HES.25-28,30-35 In addition to the imparted functions of NPs, administration route of drug-loaded NPs is also a critical issue. For example, NPs administered by intravenous injection are readily eliminated by RES during their circulation, which in turn limits their efficiency to various degrees.36,37 In view of this problem, a partial and temporary RES blockade strategy is clearly attractive. In an early study, good antitumor efficacy was achieved by employing large liposomes and small PEGylated liposomes to deliver paclitaxel.38 However, as mentioned earlier, liposomes and PEGylated liposomes have some undesirable properties and side effects.9,10,13-15,17,20,21 Therefore, safe and effective polymeric nanocarriers and their administration approach still highly need to be explored. In the current study, hydroxyethyl starch-grafted-polylactide (HES-g-PLA) copolymers were synthesized and assembled into two kinds of NPs that were used as partner nanocarriers for delivering DOX. In our design, large empty HES-g-PLA NPs were used as a RES-blocking agent to temporarily block up RES in tumor-bearing mice for a suitable period of time prior to 3


Page 5 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the administration of small DOX-loaded HES-g-PLA NPs. It was found that DOX was effectively delivered towards tumor and showed remarkably enhanced antitumor capacity in comparison to free DOX. To our knowledge, our study is the first to develop HES-g-PLA partner nanocarriers and synergistically administer them to achieve significant antitumor efficacy. The obtained results demonstrate that the HES-g-PLA partner nanocarriers have potential in clinical cancer chemotherapy. 2. MATERIALS AND METHODS 2.1. Materials. HES was procured from Wuhan HUST life Sci. & Tech. Co., Ltd (Wuhan, China), and its average weight molecular weight (Mw) and degree of molar hydroxyethyl substitution were ca.70 kD and 0.5, respectively. PLA (Mw, 5kDa) was purchased from Jinan Daigang

Biomaterials

Co.,

Ltd

(Jinan,

China).

Dicyclohexylcarbodiimide

(DCC),

dimethylamino pyridine (DMAP), doxorubicin (DOX), DiR (1,1'-dioctadecyl-3,3,3',3'tetramethyl indotricarbocyanineiodide) and dimethyl sulfoxide (DMSO) were purchased from Aladdin Inc (Shanghai, China). DMSO was purified by distillation under reduced pressure and dried with 4 Å molecular sieves prior to use. All other agents were purchased from Sinopharm, China. 2.2. Synthesis and characterization of HES-g-PLA copolymers. HES-g-PLA copolymers were synthesized via esterification reaction between terminal carboxyl groups of PLA and hydroxyl groups of HES. Briefly, HES (0.5g) was dried at 105°C for 2h, and then, dissolved in 20 mL of DMSO at 60 °C to prepare a HES solution. PLA, DCC and DMAP were dissolved in 10 mL of DMSO at a molar ratio of 1:4:2 to prepare another solution. Two kinds of solutions were mixed together and the resultant mixture was vigorously stirred at 60 °C for 24 h under protection of nitrogen. After that, the collected product was introduced into a membrane tube (MWCO: 3500) and dialyzed against deionized water for 3 days. After lyophilization, the dry product was extracted with dichloromethane in a Soxhletex extractor at 4



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 44

70 °C for at least 24 h to remove the unreacted PLA, and subsequently, dried again at 45 °C under vacuum. HES-g-PLA copolymers with varied degrees of PLA substitution were synthesized by mainly changing the ratio of HES to PLA while keeping reaction conditions constant. Based on orthogonal test, two kinds of HES-g-PLA copolymers were optimally synthesized by controlling the molar ratio of HES to PLA at 1:4 and 1:7, respectively, and they were used for subsequent assembly of HES-g-PLA NPs. FTIR spectra of HES-g-PLA copolymers were performed on a FTIR instrument (Vertex70, Bruker). 1H NMR spectra of samples were recorded on a NMR spectrometer (Ascend TM 600 MHz, Bruker) using tetramethylsilane as an internal reference. DMSO-d6 was used as solvent for NMR measurements of HES, PLA and HES-g-PLAs. Degree of PLA substitution for HES-g-PLAs, denoted as DSPLA (average PLA number per HES molecule), was determined from 1H NMR spectra of HES-g-PLAs, and calculated using a formula shown in our previous study.34 2.3. Preparation of empty HES-g-PLA nanoparticles. Empty HES-g-PLA NPs were assembled in aqueous medium. In brief, HES-g-PLA (DSPLA: 1.62; 100 mg) was added to 1 mL of ultrapure water and the resulting solution was sonicated for 10 min to prepare HES-g-PLA NPs. These empty HES-g-PLA NPs were named as ENRB NPs and used as a RES-blocking agent. 2.4. Preparation of DOX-loaded HES-g-PLA nanoparticles. DOX-loaded HES-g-PLA NPs were assembled as follows. Hydrophobic DOX was first prepared by reacting 9mg of DOX·HCl with triethylamine (Et3N, the molar ratio of DOX·HCl to Et3N was 1:3) in 2 mL of mixed solvent composed of chloroform and ethanol (chloroform:ethanol=1:1, v/v) with stirring in the dark for 2 h. In a typical procedure, HES-g-PLA (DSPLA: 0.86; 30 mg) was added to 15 mL of ultrapure water, and to this solution, hydrophobic DOX (9 mg) in 2 mL of mixed solvent (chloroform:ethanol=1:1, v/v) was added dropwise. The mixture was then sonicated for 30 min 5


Page 7 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


and the resulting emulsion was further processed using a high pressure homogenizer (600kPa, twice). Afterwards, chloroform and ethanol were removed via rotary evaporation under reduced pressure. The obtained product was introduced into a membrane tube (MWCO: 3500), and dialyzed against ultrapure water for 3 days to remove the trace amount of Et3N, Et3N·HCl and free DOX, followed by lyophilization at -50 °C. These DOX-loaded HES-g-PLA NPs were named as DOX-HPNP NPs. Dry DOX-HPNP NPs were dissolved in DMSO and the DOX extracts were detected using an UV-Vis spectrometer to determine the DOX content in DOX-HPNP NPs. The calibration curve was established using DOX/DMSO solutions with DOX concentration gradients. Drug loading (DL) was calculated using the following formula: DL(%) =[(weight of DOX in nanoparticles)/(weight of nanoparticles)]×100%

(1)

2.5. Characterization of nanoparticles. Hydrodynamic size and zeta potential (ζ) of NPs were measured by a dynamic light scattering instrument (Nano-ZS90, Malvern, UK) using ultrapure water as dispersant. Morphology of NPs was viewed using a transmission electron microscope (TEM, Tecnai G2-20). In regard to TEM sample preparation, NPs were dispersed in ultrapure water to prepare a very dilute suspension (0.01 wt%), a drop of suspension was then placed onto 300 mesh carbon-coated copper grid and dried at room temperature. NPs were negatively stained using phosphotungstic acid (2.0 wt%), and allowed to dry before TEM observation. 2.6. Cell culture and tumor model. Murine hepatoma H22 cell line and murine normal NIH 3T3 cell line were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). These cells were respectively cultured in DMEM supplemented with 10% fetal bovine serum, 100U/mL penicillin and 100µg/mL streptomycin at 37 °C in a 5% CO2 atmosphere. Two types of expended cells were suspended in PBS for the further use. Six-week old male BALB/c mice (25.7±2.1g) and adult SD rats (250-280 g) were bought 6



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 44

from the Hubei Provincial Center for Disease Control and Prevention (Wuhan, China), and used for animal experiments. They were housed in an air-conditioned atmosphere with relative humidity of 50% under natural light/dark cycle conditions, and allowed free access to standard food and water. Tumor models were established by subcutaneously injecting 0.1 mL of H22 cell suspension (1×106 cells) to the right thigh of BALB/c mice. These cell-inoculated mice were used for subsequent experiments when the average volume of resultant tumors reached a range between 100 and 120 mm3. 2.7. Cytotoxicity assay in vitro. To assess the cytotoxicity of ENRB NPs, NIH 3T3 cells were seeded in 96-well plates at a density of 5×103 cells/well and were treated with varied amounts of ENRB NPs (0-1000 µg/mL) under standard culture conditions using complete medium for 24h, 48h and 72h, respectively. Cell viability was determined using a MTT essay. The group without ENRB treatment was used as control and cell viability for this group was considered as 100%. The response of H22 cells to free DOX or DOX-HPNP NPs was also examined. H22 cells were seeded in 96-well plates (5×103 cell/well) and exposed to different amounts of DOX agents to test their viability. These wells were divided into different groups and treated with free DOX or DOX-HPNP solutions containing varied DOX equivalents of 0.01, 0.1, 1 and 10µg/mL for 24 and 48 h, respectively. Viability of H22 cells was assessed using a cell counting kit (CCK-8). 2.8. Real-time near-infrared fluorescence imaging. DiR (a near-infrared fluorescent dye) was used to substitute DOX for in vivo and ex vivo imaging. Accordingly, DiR-loaded HES-g-PLA NPs, named as DiR-HPNP NPs, were prepared using the same method applied to the preparation of DOX-HPNP NPs, and they were used for tracking the in vivo accumulation of NPs. 7


Page 9 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


H22-tumor bearing BALB/c mice were pretreated with ENRB NPs, followed by i.v. administration of DiR-HPNP NPs to find out the optimal time interval between the early injected ENRB NPs and the late injected DiR-HPNP NPs. Mice were randomly divided into 4 groups, each group containing 3 mice. Among 4 groups, mice in groups 1, 2 and 3 were all injected with 0.1 mL of ENRB solution at a dose of 400mg/kg via tail vein, and 0.1 mL of DiR-HPNP solution (30µg/kg) was then applied to mice respectively in groups 1, 2 and 3 at 0.5 h, 1.5 h and 4 h after ENRB injection in sequence. Group 4 was used as control and mice in this group received only 0.1 mL of DiR-HPNP solution. Whole-body fluorescence images of mice were recorded on a near-infrared fluorescence (NIRF) imaging instrument (Caliper IVIS Lumina II, λex=740nm, λem=780-820nm, PerkinElmer), and images were taken starting from 15 min after the DiR-HPNP injection. During the imaging period, a small amount of 3% isoflurane anesthesia was intermittently applied to mice using a nose cone tube for continuous anesthesia when they were exposed to the camera. At 24 h after DiR-HPNP injection, mice were sacrificed by cervical dislocation, and tumors as well as major organs were excised for the determination of NIRF intensity. Based on time-dependent fluorescence images and semiquantitative analysis of NIRF intensity of tumor regions, the time interval between the early injected ENRB NPs and the late injected DiR-HPNP NPs was optimized as 1.5 h. In the following studies, ENRB NPs were always used 1.5 h ahead whenever they were administered together with DOX-HPNP NPs. In addition, the volume of applied ENRB solution was 0.1 mL for mice and 1.0 mL for rats but the applied ENRB dose was the same (400 mg/kg body weight) for both mice and rats when ENRB RES-blockade was involved. Besides these, all injection solutions were prepared using PBS as solvent and injections were conducted via tail vein with the injection volume of 0.1 mL for mice and 1.0 mL for rats while applying the same dosage per body weight to them both. 2.9. Pharmacokinetic analysis. Healthy male SD rats were randomly assigned to 3 groups 8



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 44

with 3 rats per group. Rats in groups 1 and 2 were administered with free DOX and DOX-HPNP NPs, respectively. Rats in group 3 were first injected with an ENRB solution, and 1.5h later, with a DOX-HPNP solution. DOX equivalent for all rats was 4mg/kg. After predetermined time intervals (5, 15 and 30 min; 1, 2, 4, 8, 12, 24 and 48 h), counting from the last injection performed for each rat, blood was collected from retro-orbital plexus of rat eye using heparinized tubes, and centrifuged at 3500 rpm for 10 min to gain plasma samples. To each plasma sample (60µL), a mixed solvent (600 µL) containing DMSO and methanol (1:9, v/v) was added, and the mixtures were stored at -20 °C overnight. DOX extracts were analyzed using high performance liquid chromatography (HPLC) combined with an atmospheric pressure chemical ionization/mass spectrometer (APCI/MS, 1100 LC/MSD Trap, Agilent) and the instrument was run under following conditions: mobile phase, mixed solvent (methanol/ammonium acetate= 80:20, v/v); flow rate, 1.0 mL/min; injection volume, 20 µL; and detection wavelength, 563 nm. The pharmacokinetic data were analyzed using Drug and Statistic software version 2.0. 2.10. Biodistribution. 3 groups of H22-tumor bearing BALB/c mice with 3 mice in each group were used for evaluating DOX biodistribution. A free DOX solution and a DOX-HPNP solution were respectively applied to mice in groups 1 and 2. The mice in group 3 were injected with a DOX-HPNP solution at 1.5h after ENRB injection. DOX equivalent for all mice was 4mg/kg. After 12-h, counting from the last injection conducted for each mouse, major organs and tumors of mice were isolated, weighed, and homogenized with 1 mL of ultrapure water. To each homogenate, a mixed solvent (2 mL) containing DMSO and methanol (1:9, v/v) was added, and mixtures were stored at -20 °C overnight. The mixtures were then vortexed for 2 min, followed by centrifugation at 11000 rpm for 15 min to obtain DOX extracts. Extracted DOX amounts in homogenates were tested using HPLC combined with APCI/MS under the 9


Page 11 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


same running conditions mentioned earlier. DOX content in each kind of tissue was presented as microgram DOX per gram tissue. 2.11. Toxicity analysis. 4 groups of H22-tumor bearing BALB/c mice (4 mice/ group) were subjected to toxic test. Similarly, groups 1 and 2 were respectively treated with free DOX and DOX-HPNP, and group 3 was injected with DOX-HPNP at 1.5h after ENRB injection. Group 4 (control) received only saline. Equivalent DOX dose for DOX-applied mice was 4mg/kg. 4 hours after the last injection implemented for each mouse, blood was collected from the retro-orbital sinus of mice and plasma was immediately separated. Taking into account the main DOX toxic effects, levels of creatine kinase (CK) and alanine transaminase (ALT) of plasma were measured to assess the possible cardiotoxicity or hepatotoxicity, respectively. 2.12. Antitumor efficacy in tumor-bearing mice. 4 groups of H22-tumor bearing BALB/c mice with 5 mice in each group were treated with different DOX formulations to assess in vivo antitumor efficiency of DOX-HPNP NPs working with or without the ENRB partner. The treatment was started when the tumor volume reached the range between 100 and 120 mm3. Groups 1 and 2 received free DOX and DOX-HPNP NPs. The mice in group 3 were injected with a DOX-HPNP solution at 1.5 h after ENRB injection. DOX equivalent for DOX-applied mice was 4 mg/kg. Group 4 (control) received only saline. Injection for all mice was conducted every 4 days for 3 times in total, and the day for conducting the first injection was designated as day 1. Body weight of mice was measured every 2 days, and tumor volume was estimated using an empirical formula proposed in the literature.18 At the end of 13-day treatment, mice were sacrificed, and tumors were excised for their volume and weight determination. Additionally, major organs such as heart, liver, spleen, lung, and kidney, were also harvested, fixed with parafomaldehyde and sectioned into slices (5µm in thickness) for subsequent histopathological analysis using hematoxylin and eosin (H/E) staining. The H/E-staining tissue sections were 10



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 44

viewed by a pathologist. 2.13. Statistical analysis. Data were presented as mean ± standard deviation. One-way analysis of variance with post hoc test was carried out using statistical software (SPSS 15.0 for Windows) to see whether significant differences existed among the measured data, and p values less than 0.05 were considered to be statistically significant. 3. RESULTS AND DISCUSSION 3.1. Characterization of HES-g-PLA copolymers. For the sake of simplicity of description, a HES-g-PLA copolymer and different HES-g-PLA copolymers with varied degrees of PLA substitution are referred to as HES-g-PLA and HES-g-PLAs, respectively. HES-g-PLA nanoparticles will be simply called as HES-g-PLA NPs in the following text. To endow HES-g-PLAs with uniform and long PLA side-chains, HES was grafted with PLA having designated molecular weight and terminal groups via esterification reaction between carboxyl groups of PLA and hydroxyl groups of HES. A schematic diagram for the synthesis of HES-g-PLAs is presented in Figure 1, and representative FTIR spectra for HES, PLA and HES-g-PLA are presented in Figure S1. In the spectrum of HES, a wide peak at around 3450 cm-1, and two other peaks appeared at 2931 and 1649 cm-1 can be respectively ascribed to O-H stretching, C-H vibration absorption and intramolecular hydrogen bonds in HES molecules.34,39 The spectrum of PLA shows two typical peaks at 1745 and 1187 cm-1, indicating the stretching vibration of carbonyl groups and symmetric vibration of C-O-C. With regard to HES-g-PLA, a week peak at 2991 cm-1 denotes the stretching of PLA-CH3. It should be noticed that the peak at 1648 cm-1 becomes smaller for HES-g-PLA when comparing with the matched one for HES, revealing less formation of intramolecular hydrogen bonds in HES-g-PLA due to the PLA grafting. Another peak at 1753 cm-1 is attributed to the shift of the peak originally located at 1745 cm-1 in the spectrum of PLA, providing a robust evidence for the occurrence of grafting.40,41 11


Page 13 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Typical 1H NMR spectra for HES, PLA and HES-g-PLA are represented in Figure S2. In the case of HES, peaks registered in the range between 4.4 and 5.7 ppm belong to different kinds of protons, including the protons bonding to the C-1 sites of hydroglucose units (AGU) and the protons in three hydroxyl groups in AGU, no matter whether hydroxyl groups are directly attached to the glucose or to the hydroxyethyl moiety.27 With respect to PLA, the peak located at 1.47 ppm is indicative of methyl groups on PLA chains and at the chain terminals; anther peak near 5.18 ppm is assigned to -CH protons in PLA chains, and a very weak peak at 13.1 ppm is attributed to protons in terminal carboxyl groups of PLA. As for HES-g-PLA, the signal appeared at 1.5 ppm is ascribed to -CH3 protons in PLA chains, and signals detected in the range between 4.4 and 5.7 belong to HES. The original peak of PLA at around 5.18 ppm is not registered in the spectrum of HES-g-PLA, which is due to the peak overlap occurred between PLA and HES components. It is worth noting that an original weak peak at 13.1 ppm for the PLA component completely disappears, which should suggest that the terminal carboxyl groups in PLA chains have reacted with hydroxyl groups in HES to form ester bonds.42 Based on the results shown in Figures S1 and S2, it can be concluded that PLA has been successfully grafted on to the HES backbone. 3.2. Characterization of HES-g-PLA nanoparticles. The size of NPs is known to be a key factor that can significantly influence their in vivo performance.43 Some studies have suggested that NPs with sizes in the range between 40 and 200nm are likely to accumulate in tumor via enhanced permeability and retention (EPR) effect; and NPs having larger sizes (about 300 nm or more) would be more easily caught by RES in liver and spleen.23,43-46 In the present study, empty HES-g-PLA NPs assigned for blocking up RES, therefore, should be larger than 300 nm for the effective RES-blockade, and on the other hand, DOX-loaded HES-g-PLA NPs have to be small in order to achieve high intratumoral accumulation. In addition to controlling their sizes, HES-g-PLA NPs also need to have proper structures 12



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 44

and properties that allow them to well suit for RES-blockade and drug delivery, respectively. Effects of HES and PLA on HES-g-PLAs and HES-g-PLA NPs were thus carefully examined. Investigations into the HES component indicated that branched HES was able to endow the resultant HES-g-PLA NPs with a notably higher ability to resist the α-amylase-mediated degradation as compared to linear HES. In addition, outcomes from our comparative experiments confirmed that the molecular weight of branched HES would significantly regulate the sizes and anti-degradation properties of HES-g-PLA NPs. Based on many trails, the branched HES containing around 95% of amylopectin and having its molecular weight of ca.70 kD was selected as a starting material for the synthesis of HES-g-PLAs. The effects of molecular weight and substitution degree of PLA on HES-g-PLA NPs were also studied. The preliminary experimental results revealed that the molecular weight and substitution degree of PLA would notably modulate the size, DL and degradation rate of HES-g-PLA NPs. Considering that both HES and PLA components have to work together in a coordinated manner to control the properties of resulting HES-g-PLA NPs, the molecular weight and substitution degree of PLA were thus optimized through orthogonal test on condition that the mentioned parameters for branched HES was included in test. By controlling the molecular weight of PLA at an optimal value of 5000, two kinds of desirable HES-g-PLAs (DSPLA: 0.82 and 1.62) were synthesized by changing the molar ratio of HES to PLA while keeping the reaction conditions constant. As described in the experimental section, two kinds of HES-g-PLA NPs were assembled in aqueous media with the help of ultrasonic dispersion. DSPLA of HES-g-PLAs and concentrations of aqueous HES-g-PLA solutions were found to exert considerable impacts on sizes and properties of resultant HES-g-PLA NPs when this ultrasonic processing method was utilized. Based on many comparative trials, HES-g-PLA with DSPLA of 1.62 was selected to assemble large empty HES-g-PLA NPs, i.e. ENRB NPs, and parameters for ENRB NPs are 13


Page 15 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


listed in Table 1. ENRB NPs had a wide size-distribution with a mean size of around 730 nm. The wide size-distribution can be ascribed to the presently used dispersion preparation technique, which usually results in wide size-distributed particles.23 As mentioned earlier, larger NPs with sizes larger than 300nm would be likely detained by RES in liver and spleen.23,43,44 In the present situation, ENRB NPs had their mean size of around 730 nm, and their lower size limit was 300 nm, meaning that they should be suitable for the RES-blocking usage from the perspective of size account. Table 1 also exhibits that ENRB NPs were negatively charged with a zeta potential of around -5.2 mV, suggesting that ENRB NPs would tend to translocate into RES-rich organs during their in vivo circulation due to their negatively charged surface.23,43,44,47,48 Unlike empty HES-g-PLA NPs, DOX-loaded HES-g-PLA NPs have to be small in size to enhance the DOX intratumoral accumulation. In contrast to ENRB NPs, relatively smaller DOX-loaded NPs could be achieved by using the HES-g-PLA having the DSPLA of 0.86 as precursor, and employing a combined processing technique composed of prior emulsification and the followed high-pressure homogenization. Under optimized processing conditions, DOX-loaded HES-g-PLA NPs, namely, DOX-HPNP NPs, were successfully assembled and relevant results are illustrated in Figure 2. Figure 2(A) exhibits that these DOX-HPNP NPs had good sphericity and their size was estimated to range from several tens of nanometers to around 100 nm. Figure 2(B) shows that the size of hydrated DOX-HPNP NPs had an approximate Gaussian distribution over a range between about 40 nm and 300 nm. Several sets of DOX-HPNP NPs were measured and their parameters are summarized in Table 2. Table 2 denotes that DOX-HPNP NPs had a mean size of about 130 nm with a slightly positive ζ potential of 1.6 mV. Generally speaking, a smaller mean size for these DOX-HPNP NPs might be better for enhancing the DOX delivery towards tumors via EPR effect. Nevertheless, in the 14



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 44

present case, it is extremely difficult to further reduce the mean size of DOX-HPNP NPs because the branched HES molecules in hydrated state are in a rough shape of microspheres with their size larger than 10 nm, and would impede the formation of small-size NPs.26-28 Table 2 also shows that average DL for DOX-HPNP NPs was about 8 wt%. Although a higher DL can be achieved following the same assembly method, DL for these NPs was not further increased because our observations indicate that DOX-HPNP NPs with a DL higher than 10wt% could possibly result in aggregation during their long-term storage. As a matter of fact, this initial DOX load should be high enough for the in vivo DOX-HPNP use owing to the merits of two precursors used for the synthesis of HES-g-PLA. It is known that HES, as a plasma substitute, can be used at a large daily dose.29 On the other hand, PLA, as a fully biodegradable and biocompatible polymer, accounts for only a low percentage of HES-g-PLA. Therefore, DOX dosage can be facilely regulated by changing the DOX-HPNP amount within a rational and safe range. Therefore, the average DL for DOX-HPNP NPs was selected as 8.2wt% for all subsequent studies. The physical stability of NPs is an important issue in relation to their performance.23,46 DOX-HPNP NPs were thus stored in PBS at 25°C for one week to test whether their size significantly changed with storage time. No precipitation was viewed for the stored DOX-HPNP NPs during 7-day storage based on visual observations. Time-varying sizes for the stored DOX-HPNP NPs are shown in Figure 2(C). It can be seen that the size of hydrated DOX-HPNP NPs changed slightly without significant differences over 7 days. A representative TEM image for DOX-HPNP NPs stored in PBS for 7 days is presented in Figure 2(D). By comparing the image shown in Figure 2(D) with that presented in Figure 2(A), it can be concluded that they show similar shape and size. These results demonstrate that DOX-HPNP NPs are stable in PBS. 3.3. In vitro cytotoxicity assessment. In vitro cytotoxicity of ENRB NPs was tested using 15


Page 17 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


NIH 3T3 cells and relevant results are illustrated in Figure 3(A). The viability of cells was higher than 90 % after 24-h incubation without significant differences when the applied ENRB dose changed from 250 to 1000 µg/mL; after being incubated for 48 h, a similar situation was registered; and viability of cells became slightly lower but still reached around 90% or higher when the incubation time was extended up to 72h. Results shown in Figure 3(A) verify that ENRB NPs are nearly nontoxic to normal cells in view of large ENRB dosage and high viability of NIH 3T3 cells. The effects of free DOX and DOX-HPNP NPs on the viability of H22 cells were also examined and results are elucidated in Figures 3(B) and 3(C). After 24-h incubation, cell viability decreased from ca. 98% to around 24% and 34%, respectively corresponding to free DOX and DOX-HPNP, when the DOX equivalent was altered from 0.01 to 10 µg/mL. Free DOX showed much higher (p

Enhancing Doxorubicin Delivery toward Tumor by ... - ACS Publications

Recommend Documents