Development of Novel Lignin-Based Targeted ... - ACS Publications

Subscriber access provided by Warwick University Library

Controlled Release and Delivery Systems

Development of novel lignin-based targeted polymeric nanoparticle platform for efficient delivery of anticancer drugs Kefeng Liu, Dan Zheng, Hantian Lei, Jing Liu, Jiandu Lei, Luying Wang, and Xingyuan Ma ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00260 • Publication Date (Web): 27 Mar 2018 Downloaded from http://pubs.acs.org on April 4, 2018

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 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 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.

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 28 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 Biomaterials Science & Engineering

Development of novel lignin-based targeted polymeric nanoparticle platform for efficient delivery of anticancer drugs Kefeng Liua, Dan Zhenga, Hantian Leia, Jing Liua, Jiandu Leia*, Luying Wanga*and Xingyuan Mab* a

Beijing Key Laboratory of Lignocellulosic Chemistry, Beijing Forestry University, Beijing, 100083, P. R. China

b

State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237, P. R. China

ACS Paragon Plus Environment

1

ACS Biomaterials Science & Engineering 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 28

ABSTRACT

The clinical applications of natural anticancer drugs are being restricted by poor water solubility, fast clearance in the circulation, lack of targeting to tumor cells and poor tissue penetration. To address these problems, in this study, we developed a novel lignin-based targeted polymeric nanoparticles (NPs) platform, folic acid-polyethylene glycol-alkaline lignin conjugates (FAPEG-AL), via self-assembly for delivery of anticancer drug (hydroxyl camptothecin, HCPT). These lignin-based nanoparticles had moderate particle size (~150 nm) with a narrow size distribution (PDI < 0.1), exhibited excellent biocompatibility, high drug loading efficiency (~24.2 wt% of HCPT), prolonged blood circulation time (~7-fold of free HCPT) and enhanced cellular uptake (~5-fold of free HCPT). Besides, the drug biodistribution study confirmed preferred accumulation of FA-PEG-AL/HCPT NPs in tumor tissue. Subsequent tumor xenograft test revealed superior tumor suppression efficacy and reduced side effects of FA-PEG-AL/HCPT NPs compared with free HCPT. Therefore, the prepared lignin-based FA-PEG-AL/HCPT NPs would be a promising candidate for anticancer drugs delivery.

KEYWORDS : polymeric nanoparticle, drug delivery, lignin, renewable biopolymer, selfassembly, hydroxyl camptothecin


2

Page 3 of 28 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


1. Introduction According to World Health Organization (WHO), malignant cancer is the leading cause of death in humans and caused about 9 million death annually.1 Although great progress has been made in cancer treatment, chemotherapy remains the mainstay of treatment at this stage. However, the clinical use of natural anticancer drug has not always achieved desired therapeutic effect due to their inherent drawbacks, such as poor water solubility, fast clearance in the circulation, lack of targeting to tumor cells and poor tissue permeability.2 Therefore, the development of a safe and convenient drug delivery system for these natural anticancer drugs delivery is urgently needed. Fortunately, the development of nanotechnology brings new ideas for the treatment of cancer. Polymeric nanoparticles (NPs) have recently attracted significant attention because of their great potential for drug delivery.3-7 As a versatile nanocarrier, polymeric NPs exhibit high drug loading efficiency, prolonged half-life in the bloodstream, sustained drug release behavior, good bioavailability and tissue permeability. Besides, polymeric NPs can preferential accumulate in the tumor site owe to the enhanced permeability and retention (EPR) effect.8 Although promising, it is still a challenge to prepare polymeric NPs with good a good safety profile . Recently, the natural biopolymers have attracted significant interest in biomedical application due to their good biocompatibility and biodegradability.9 To date, various natural biopolymers have been used to prepare nanocarriers for drug delivery. For example, Ernsting et al. used carboxymethyl cellulose to prepare nanoparticles for the delivery of docetaxel , and performed in vitro and in vivo efficacy assays.10-12 Lv et al. used carboxymethyl chitosan to prepare nanoparticles for the delivery of insoluble drug paclitaxel, which had demonstrated superior anticancer efficacy compared with free paclitaxel.13-14 Simi et al. used starch to prepare cross-


3


Page 4 of 28

linked nanoparticles for drug delivery.15 Lignin, as the second most abundant and renewable natural biopolymer, also has received numerous interest from researchers. Some of the current commercial applications of lignin include its use as a filler, additive, binder, dispersant, adsorbent or surfactant.16-21 In fact, lignin has many advantages, such as adequate reactive groups that enable chemical modifications, biodegradability, biocompatibility and low toxicity, all this making it an ideal candidate for drug delivery.22 In some previous researches, the use of lignin for drug delivery has been reported.23-26 However, the preparation of lignin-based nanocarriers with long circulation time in bloodstream and high binding affinity toward tumor cells remains challenging. In this work, a novel lignin-based polymeric NPs platform with high binding affinity toward tumor cells and long circulation time in bloodstream was successfully developed via selfassembly for efficient delivery of anticancer drug HCPT. The particle size and distribution, in vitro stability and drug release profile, cellular uptake, in vivo biodistribution and circulation half-life of prepared FA-PEG-AL/HCPT NPs were characterized to evaluate its potential as a nanocarrier for drug delivery. Additionally, in vitro cytotoxicity of FA-PEG-AL/HCPT NPs was measured in LLC and L929 cells. Finally, the LLC-tumor bearing mouse model was established for in vivo anticancer effect evaluation.


4

Page 5 of 28 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


2. Experimental details 2.1 Materials and regents Alkaline lignin (AL) was purchased from Henan Qiangxing Chmical Co., Ltd. (Henan, China). 10-Hydroxy camptothecin (HCPT) was received from Shanghai Yihe Biological Co., Ltd (Shanghai, China). Amine polyethylene glycol carboxyl (NH2-PEG-COOH, Mw 2000) and methoxy polyethylene glycol carboxyl (M-PEG-COOH, Mw 2000) were supplied by Jenkem Technology (Beijing, China). Folic acid (FA), N-hydroxysuccinimide ester (NHS), 4dimethylaminopyridine

(DMAP)

and

1-Ethyl-3-(3-dimethylaminopropyl)

carbodiimide

hydrochloride (EDC . HCL) were purchased from Sigma-Aldrich. Lewis lung carcinoma (LLC) cells and L929 cell line were received from Peking University Health Science Center (Beijing, China) and cultured in Dulbecco’s modified eagle’s medium (DMEM) and RPMI-1640 medium containing 10% fetal bovine serum (FBS), respectively. All cells were incubated in a humidified atmosphere of 5% CO2 incubator at 37 °C. Male C57BL/6 mice (~6 weeks old) were supplied by Institute of Genetics and Developmental Biology (Beijing, China). Animal protocols followed the institutional ethics committee regulations and guidelines on animal welfare (Peking University). 2.2 Synthesis of FA-PEG-AL conjugates The FA-PEG conjugates were synthesized as reported previously.27 Briefly, FA (0.44 g) was dissolved into 15 mL of anhydrous dimethyl sulfoxide (DMSO), EDC (0.23 g) and NHS (0.18g) were subsequently added and stirred at 25 °C for 4 h. NH2-PEG-COOH (2.0 g) was then added with gentle stirring. The crude FA-PEG conjugates were obtained after reaction for 48 h and further purified by dialysis (MWCO 2000 Da).


5


Page 6 of 28

Figure 1. Schematic of self-assembly process of FA-PEG-AL/HCPT NPs and synthetic process of FA-PEG-AL conjugates. The FA-PEG-AL conjugates were synthesized via a simple esterification reaction through the carboxyl of FA-PEG and hydroxyl of AL. Briefly, FA-PEG (0.128 g) and AL (0.50 g) were dissolved into tetrahydrofuran (15 mL). Followed, EDC (0.12 g) and DMAP (0.14 g) were added and stirred at 25 °C for overnight. The crude FA-PEG-AL conjugates were collected after rotary evaporation, and further precipitated by excess cold ethyl ether and purified by dialysis (MWCO 2000 Da). The PEG-AL conjugates were synthesized using the similar method by replacing FAPEG with M-PEG-COOH. The successful synthesis was confirmed by 1H-NMR. 2.3 Fabrication of FA-PEG-AL/HCPT NPs FA-PEG-AL/HCPT NPs were prepared by a nanoprecipitation method.28-29 Briefly, free HCPT (4 mg) and FA-PEG-AL conjugates (8 mg) were dissolved in 5 mL of

tetrahydrofuran.

Followed, the deionized water was added to the mixture using a burette with gentle stirring until


6

Page 7 of 28 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 final water content reached 75 wt%. After stirring for 30 min, FA-PEG-AL/HCPT NPs solution was obtained. All other HCPT-loaded NPs were prepared by the similar method. 2.4 Characterization of HCPT-loaded NPs The size, ζ-potential and polydispersity index (PDI) of the prepared nanoparticles (AL, AL/HCPT, PEG-AL/HCPT and FA-PEG-AL/HCPT) were measured using a Zeta Sizer Nano Series (Malvern Instruments, UK). The morphology of the FA-PEG-AL/HCPT NPs were observed using transmission electron microscope (TEM, JEM-1400, Japan) and scanning electron microscope (SEM, JSM-6700F, Japan). The in vitro stability of various HCPT-loaded NPs were measured in PBS solution (pH 7.4). At selected time points, samples in PBS solution were withdraw and particle size and ζ- potential were determined according to the above method. The HCPT concentrations were measured by high performance liquid chromatography (HPLC) equipment with a UV detection at absorbance of 254 nm, it uses a C18 column (5µm, 4.6 × 250 mm) with acetonitrile-0.1% phosphoric acid aqueous solution as mobile phase (27 : 73, 1.0 mL/min) at 25 °C. The calibration curves were generated using known concentrations of HCPT. The drug loading efficiency (DLE) and encapsulation efficiency (EE) were calculated as follows: DLE (wt%) = (weight of HCPT in NPs/weight of HCPT-loaded NPs) × 100% EE (%) = (weight of HCPT in NPs/weight of HCPT added initially) × 100% 2.5 In vitro drug release The drug release behaviors of HCPT from HCPT-loaded NPs were measured using a dialysis method.30 Briefly, 10.0 mg of different HCPT-loaded NPs (AL/HCPT, PEG-AL/HCPT and FAPEG-AL/HCPT) were suspended in 10 mL of PBS solution (pH 7.4), and then poured into a dialysis bag with 1000 Da MWCO. Followed, the dialysis bag was immersed in PBS buffer (pH


7


Page 8 of 28

7.4, 200 ml). At determined time points, PBS medium (2 mL) was withdrawn and replaced with the equal fresh PBS, and HCPT concentrations were measured by HPLC. 2.6 In vitro cellular uptake LLC cells were cultured on 12-well plates. After incubated for 24 h, cells were treated with free HCPT and HCPT-loaded NPs (AL/HCPT, PEG-AL/HCPT and FA-PEG-AL/HCPT) for 4 h. Followed, cells were washed with PBS (three times), resuspended with PBS, and detected by flow cytometer (Beckman Coulter, USA). Besides, folic acid receptor (FAR)-mediated specific cellular uptake was proved by adding excess free FA to FA-PEG-AL/HCPT NPs well. To visualize HCPT internalization, LLC cells were cultured onto sterile 6-well plates and incubated for 24 h. Free HCPT and FA-PEG-AL/HCPT NPs were then added and treated for 4 h. Followed, cells rinsed three times in PBS, fixed with 4% paraformaldehyde for 20 min. The cell nuclei was stained with DAPI. Images were taken with a confocal laser scanning microscopy (CLSM, PerkinElmer). 2.7 Cytotoxicity study LLC and L929 cells were cultured in 96-well plates (2 × 104 LLC cells/well, 1 × 104 L929 cells/well) with 180 µL medium. After incubation for 24 h, cells were treated with 20 µL of serial dilution of formulations for different times. Subsequently, CCK-8 solution (20 µL) was added to cells. After 4 h, the absorbance was measured at 450 nm using a Tecan M200 microplate spectrophotometer. To further compare the potency of different formulations, the values of half maximal inhibitory concentration (IC50) were estimated from cell viability curves (Figure 4c and d) and summarized in Table 2.


8

Page 9 of 28 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


2.8 Blood circulation study The normal C57BL/6 mice were treated with free HCPT or HCPT-loaded NPs (AL/HCPT, PEG-AL/HCPT and FA-PEG-AL/HCPT) via tail vein. Then, vein blood samples (100 µL) were collected at predetermined time intervals and immediately centrifuged. The HCPT concentrations of different formulations in serum were detected by HPLC. 2.9 In vivo biodistribution study The biodistribution study was performed on LLC-tumor bearing C57BL/6 mice. Mice were treated with free HCPT and HCPT-loaded NPs (AL/HCPT, PEG-AL/HCPT and FA-PEGAL/HCPT) via tail vein. After 48 h, the mice were sacrificed. The major organs and tumor tissues were collected. The HCPT distribution in organs and tumor tissues was measured by HPLC. 2.10 In vivo tumor suppression study LLC cells (2×106) were inoculated subcutaneously to the mice at the right abdomen. At three weeks postinoculation (the tumor volume about 2000 mm3), mice were divided into five groups randomly and treated with PBS, HCPT (10 mg/kg), AL/HCPT NPs, PEG-AL/HCPT NPs or FAPEG-AL/HCPT NPs (10 mg/kg of HCPT) every two days for five times via tail vein for each group. To evaluate the therapeutic effect, tumor volume was measured by a caliper and calculated as: (longest diameter × shortest diameter × shortest diameter)/2. The relative tumor volume (RTV) was calculated as: (tumor volume of treatment time/tumor volume of initial time). The tumor growth inhibition (TGI) was calculated as below : (1 − RTV of treatment group/RTV of control group) × 100%. The acute toxicity signs including the body weight and behavior of mice were also monitored.


9


Page 10 of 28

2.11 Detection of the hypersensitivity The hypersensitivity of different drug formulations (PBS, free HCPT, AL/HCPT NPs, PEGAL/HCPT NPs and FA-PEG-AL/HCPT NPs) were performed in five groups of LLC-tumor bearing C57BL/6 mice. The drug formulations were administrated via tail vein at HCPT dose of 10 mg/kg body weight every two days. After treatment for 12 days, vein blood was harvested and IgE level was measured by IgE ELISA (R&D systems). Besides, the hematocyte numbers including platelets (PLT) and white blood cell (WBC) were detected by a hematology analyzer (MEK-7222K, Nihon Kohden Celltac E) after the last treatment. 2.12 Statistical analysis All data were expressed as means ± standard errors (SD). Statistical significance was determined using a student’s t-test or one-way ANOVA and P < 0.05 (*) was considered statistical significant. 3. Results and discussion 3.1 Synthesis of FA-PEG-AL conjugates The schematic synthesis of FA-PEG and FA-PEG-AL conjugates were shown in Figure. 1. The representative 1H-NMR spectrum were characterized to confirm the successful synthesis. As shown in Figure 2a-c, the signals which was appeared at δ = 8.64, 8.10, 7.64, 6.91, 6.63, 4.48, 4.32, 2.31, 1.23 ppm (FA), δ = 3.50 ppm (PEG), and δ = 7.75, 7.02, 3.62 ppm (AL) confirmed the synthesis of FA-PEG-AL conjugates. Because of the amido bonds formed, the signal at δ = 2.31 of FA moved to δ = 2.29, confirmed the successful synthesis of FA-PEG conjugates.


10

Page 11 of 28 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


Figure 2. 1H-NMR spectra of FA (a), FA-PEG conjugates (b), and FA-PEG-AL conjugates (c) in DMSO-d6. 3.2 Preparation and characterizations of FA-PEG-AL/HCPT NPs In order to efficient delivery of anticancer drugs, AL, PEG-AL and FA-PEG-AL conjugates were used to prepare nanoparticles via self-assembly, and HCPT was encapsulated as model drug. As shown in Table 1, all the HCPT-loaded NPs exhibited high DLE and EE, which is favorable for improving intratumoral drug concentrations. The main physicochemical characteristics of the prepared HCPT-loaded NPs including particle size, ζ- potential and PDI were characterized and the detailed results were shown in Table 1. The TEM and SEM images (Figure 3a and b) showed that the FA-PEG-AL/HCPT NPs exhibited regularly spherical. As shown in Figure 3c, the particle sizes determined by size analyzer were in good agreement with the TEM and SEM images. The ζ-potential is an important factor to assess the stability and intracellular uptake of nanoparticles. As shown in Figure 3d, the blank and AL/HCPT NPs exhibited negative charges.


11


Page 12 of 28

After be modified with PEG chain, the ζ-potential of PEG-AL/HCPT and FA-PEG-AL/HCPT were close to neutrality, indicating that the outer coating of PEG chains could shield the negative charges presented on the AL/HCPT NPs surface. This shielding behavior would be beneficial to reduce the charge repulsion between AL/HCPT NPs and the cell membrane, and thus enhanced the cellular uptake of nanoparticles. The stability is another important parameter for nanoparticles. Considering that particle size and ζ-potential have direct relevance with stability, we thus tested the stability of nanoparticles in corresponding aspects.31 As illustrated Figure 3d and e, the size and ζ-potential of nanoparticles had no obvious change within 96 h, reflecting the good stability, which is beneficial to following investigations. The release profiles of HCPT from HCPT-loaded NPs (AL/HCPT, PEG-AL/HCPT and FAPEG-AL/HCPT) were tested in vitro. As presented in the Figure 3f, there was no significant burst release behavior among all the three HCPT-loaded NPs. Interestingly, compared with AL/HCPT NPs, the release rate was obviously increased once modified with PEG, which might attribute to the improvement of hydrophilicity via PEG chain. After incubation for 192 h, the cumulative amount of HCPT released from FA-PEG-AL/HCPT NPs was above 80%, which would be beneficial to enhance the anticancer effect. Table 1. Physicochemical characteristics of blank and different HCPT-loaded nanoparticles Compound

DLE (wt%)

EE (%)

Size (nm)

ζ-potential (mV)

PDI

AL NPs

-

-

116.4 ± 7.6

-40.2 ± 4.8

0.061

AL/HCPT NPs

28.4 ± 3.7

80.4 ± 4.2

135.1 ± 7.4

-35.5 ± 4.2

0.049

PEG-AL/HCPT NPs

26.1± 2.9

76.5 ± 2.4

148.2 ± 8.8

3.4 ± 0.8

0.064

FA-PEG-AL/HCPT NPs

24.2 ± 3.1

74.4 ± 2.8

152.6 ± 9.6

2.1 ± 0.4

0.058


12

Page 13 of 28 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


Figure 3. TEM (a), SEM (b) and size distribution (c) of FA-PEG-AL/HCPT NPs. Stability of size (d), and ζ-potential (e) of different nanoparticles for 96 h of storage at 37 °C. HCPT release profiles from different nanoparticles (f). 3.3 Cytotoxicity study The in vitro cytotoxicity of HCPT, AL NPs and HCPT-loaded NPs (AL/HCPT, PEGAL/HCPT and FA-PEG-AL/HCPT) were measured toward LLC and L929 cells by a CCK-8 assay. As shown in Figure 4a and b, the cytotoxic effect were related with the treatment time, and the FA-PEG-AL/HCPT NPs exhibited highest cytotoxicity both in LLC and L929 cells. As shown in Figure 4c and d, the AL NPs exhibited very low cytotoxicity, reflecting its good biocompatibility. Free HCPT showed slight cytotoxicity effect. As expected, the cytotoxicity effect were increased once HCPT was loaded in nanoparticles. Interestingly, FA-PEG-AL/HCPT NPs showed preferential cytotoxicity effect toward LLC cells due to the FAR- mediated specific endocytosis. Besides, IC50 values of different drug formulations were determined from cell viability curves (Figure 4c and d) and summarized in Table 2.


13


Page 14 of 28

Figure 4. Cell viability of LLC (a) and L929 (b) cells treated with HCPT and HCPT-loaded NPs (equivalent to free HCPT) for 24,48 and 72 h. Cell viability of LLC (c) and L929 (d) cells treated with different concentrations of formulations. Table 2. In vitro cytotoxicity analysis (IC50, µg/mL) Compound

LLC cells

L929 cells

HCPT

0.51 ± 0.14

0.63 ± 0.17

AL/HCPT NPs

0.44 ± 0.12

0.52 ± 0.12

PEG-AL/HCPT NPs

0.32 ± 0.09

0.45 ± 0.14

FA-PEG-AL/HCPT NPs

0.12 ± 0.04

0.38 ± 0.10


14

Page 15 of 28 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


3.4 In vitro cellular uptake In order to determine whether our nanoparticles internalized into the tumor cells, we performed flow cytometry and CLSM experiments. As shown in Figure 5a and b, the internalization amount of free HCPT was very little, which was confirmed by the mean fluorescence intensity (MFI) in LLC cells. On the contrary, HCPT was more extensively internalized once encapsulated into PEG-AL NPs. The possible reason is that free HCPT internalized into the cell by a passive diffusion manner, which was easily hindered by P-glycoprotein on the cell membrane.32 Traditionally, nanoparticles were internalized into cells by an endocytic pathway, thereby avoiding the effect of P-glycoprotein pumps.33 In addition, the cellular uptake was further promoted by FA conjugation owe to the FAR-mediated specific endocytosis, which was confirmed by its inhibition by adding excess free FA (10-fold). The results revealed that the cells treated by excess free FA and FA-PEG-AL/HCPT NPs showed negligible increase in the

Figure 5. Cellular uptake of different formulations (a and b). Confocal images of LLC cells incubated with free HCPT (c) and FA-PEG-AL/HCPT NPs (d).


15


Page 16 of 28

fluorescence levels compared with PEG-AL/HCPT NPs, which should be attributed to that the binding sites of FA-PEG-AL/HCPT NPs were occupied by the excess free FA. The CLSM imaging results were in good agreement with flow cytometry. As shown in Figure 5c and d, FAPEG-AL/HCPT NPs treatment group exhibited strong fluorescent signal, but became weak in free HCPT group, suggesting the favorable HCPT delivery of FA-PEG-AL NPs. 3.5 In vivo blood circulation study The blood circulation time of different formulations were performed in normal C57BL/6 mice. As presented in Figure 6a and b, free HCPT displayed rapid plasma elimination with half-life was only 1.6 h. However, the half-life of HCPT was remarkable increased once encapsulated in AL NPs (~4.1 h). Besides, the half-lives were further increased by modified with PEG chain (~10.2 h for PEG-AL/HCPT, ~11.8 h for FA-PEG-AL/HCPT). The possible reason is that PEG chain could reducing the nonspecific interaction of nanoparticles with reticuloendothelial system.34 The above results demonstrated that the prepared FA-PEG-AL/HCPT NPs were an excellent nanocarrier to prolong the half-life of anticancer drugs in blood. 3.6 In vivo biodistribution study In order to ascertain whether our nanoparticles could accumulate in tumor site, we performed the in vivo biodistribution study in LLC-tumor bearing mice. As presented in Figure 6c, a very few amount of HCPT was accumulate in tumor site in group of free HCPT owe to its rapid metabolism. However, the HCPT accumulation in tumor site was significantly increased in nanoparticle groups due to the EPR effect.35 The FA-PEG-AL/HCPT NPs exhibited 3.5 times and 1.8 times higher accumulation compared with AL/HCPT and PEG-AL/HCPT NPs owe to the coating of PEG and FA. Besides, the HCPT levels in major organs, including heart, liver, spleen, lung, and kidney were significantly lower. These results demonstrated that the


16

Page 17 of 28 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


Figure 6. Blood circulation curves (a) and half-life (b) of different drug formulations. Tissue distribution treated with the different drug formulations for 48 h (c). FA-PEG-AL/HCPT NPs could effectively accumulated in tumor tissues and cause less damage to normal organs. 3.7 In vivo tumor suppression study The excellent anticancer efficacy of FA-PEG-AL/HCPT NPs described above gave us great motivation to further explore its in vivo performance. As shown in Figure 7b and c, administration of FA-PEG-AL/HCPT NPs significantly suppressed the tumor growth and extended the survival time of mice compared with other groups. At the day of 24, the tumor volume treated with FA-PEG-AL/HCPT NPs was only 19.7% compared with group of PBS, which was 4.2 times, 2.8 times and 1.9 times smaller than free HCPT, AL/HCPT and PEGAL/HCPT NPs group, respectively. Besides, there was no noticeable body weight change of mice in FA-PEG-AL/HCPT NPs group, suggesting its good in vivo safety profile.


17


Page 18 of 28

Figure 7. Schematic of tumor suppression process (a). Tumor suppression (b), survival rates (c) and body weight (d) of LLC-tumor bearing mice treated with different drug formulations. Table 3. The anticancer efficacy of different groups. Compound

TV (mm3)a

RTVa

TGI (%)a

Cures (%)a

PBS

4420 ± 1260

34.3 ± 14.8

-

0

HCPT

3696 ± 1026

28.4 ± 11.2

17.2 ± 9.8

33.3

AL/HCPT NPs

2529 ± 684

18.6 ± 7.4

45.8 ± 19.2

50

PEG-AL/HCPT NPs

1660 ± 547

12.8 ± 5.8

62.7 ± 29.7

66.7

FA-PEG-AL/HCPT NPs

878 ± 298

6.7 ± 3.1

80.5 ± 33.4

83.3

a

TV, RTV, TGI, and cures were determined at day of 24. 3.8 Detection of the hypersensitivity Despite the potent therapeutic effect of FA-PEG-AL/HCPT NPs had been proved, the side

effects were still unclear. Considering the fact that hypersensitivity reactions (usually type-1) is the most critical side effect for many natural anticancer drugs, we thus selected IgE level (an indicator for type-1 hypersensitivity response) for detection of hypersensitivity responses.36 As presented in Figure 8a, administration of free HCPT caused higher IgE level over PBS group,


18

Page 19 of 28 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


Figure 8. Detection of side effects of different drug formulations: IgE level (a), relative ratios of WBC (b) and PLT (c). which might attribute to the water insolubility of HCPT. On the contrary, the IgE level in nanoparticle groups showed slight change, reflecting the reduced hypersensitivity reactions of nanoparticle formulations. To explore the hematotoxicity, the WBC and PLT counts of mice were harvested by a hematology analyzer (Figure 8b and c). For free HCPT group, the WBC and PLT counts were remarkably decreased compared with PBS group. As expected, the WBC and PLT counts in nanoparticle groups fluctuate in an acceptable range, suggesting that our nanoparticle formulations induced no severe hematotoxicity to treated mice. 4. Conclusion In conclusion, we have successfully developed a novel lignin-based targeted polymeric NPs (FA-PEG-AL) for efficient delivery of anticancer drug HCPT. The moderate particle size, high drug loading efficiency, robust stability, efficient delivering capacity and good biocompatibility of FA-PEG-AL/HCPT NPs revealed its promising potential for delivery of anticancer drugs. Besides, this delivery system was capable of improving the blood circulation time and enhancing cellular uptake owe to the outer coating of PEG chain and target molecular FA. Furthermore, this formulation could accumulating preferentially in tumor site by EPR effect, which was incomparable with the free HCPT. Because of these benefits, FA-PEG-AL/HCPT NPs could


19


Page 20 of 28

remarkably inhibited tumor growth and would be an efficient drug delivery system in clinical application. Corresponding Author *E-mail: [email protected]; [email protected]; [email protected] ACKNOWLEDGMENTS This study was supported by the National Natural Science Foundation of China (No.21406013; 21576029) and Beijing college students' innovation project (S201710022045). REFERENCES 1.

Siegel, R. L.; Miller, K. D.; Jemal, A., Cancer statistics, 2016. Ca A Cancer Journal for

Clinicians 2016, 66 (1), 7. DOI: 10.3322/caac.21332 2.

Blagosklonny; Mikhail, V., Overcoming limitations of natural anticancer drugs by

combining with artificial agents. Trends in Pharmacological Sciences 2005, 26 (2), 77-81. DOI: 10.1016/j.tips.2004.12.002 3.

Silva, G. A., Introduction to nanotechnology and its applications to medicine. Surgical

Neurology 2004, 61 (3), 216-20. DOI: 10.1016/j.surneu.2003.09.036 4.

Chan, J. M.; Valencia, P. M.; Zhang, L.; Langer, R.; Farokhzad, O. C., Polymeric

nanoparticles for drug delivery. Humana Press: 2010; p 163-75. DOI: 10.1007/978-1-60761609-2_11 5.

Kumari, A.; Yadav, S. K.; Yadav, S. C., Biodegradable polymeric nanoparticles based

drug delivery systems. Colloids & Surfaces B Biointerfaces 2010, 75 (1), 1. DOI: 10.1016/j.colsurfb.2009.09.001


20

Page 21 of 28 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


6.

Soppimath, K. S.; Aminabhavi, T. M.; Kulkarni, A. R.; Rudzinski, W. E., Biodegradable

polymeric nanoparticles as drug delivery devices. Journal of Controlled Release Official Journal of the Controlled Release Society 2001, 70 (1–2), 1-20. DOI: 10.1016/S0168-3659(00)00339-4 7.

Liu, K. F.; Liu, Y. X.; Dai, L.; Li, C. X.; Wang, L.; Liu, J.; Lei, J. D., A novel self-

assembled pH-sensitive targeted nanoparticle platform based on antibody–4arm-polyethylene glycol–pterostilbene conjugates for co-delivery of anticancer drugs. Journal of Materials Chemistry B 2017, 6 (4). DOI: 10.1039/c7tb02485a 8.

Maeda, H.; Bharate, G. Y.; Daruwalla, J., Polymeric drugs for efficient tumor-targeted

drug delivery based on EPR-effect. European Journal of Pharmaceutics & Biopharmaceutics Official Journal of Arbeitsgemeinschaft Fur Pharmazeutische Verfahrenstechnik E V 2009, 71 (3), 409-419. DOI: 10.1016/j.ejpb.2008.11.010 9.

Nair, L. S.; Laurencin, C. T., Biodegradable polymers as biomaterials. Progress in

Polymer Science 2007, 32 (8), 762-798. DOI: 10.1016/j.progpolymsci.2007.05.017 10. Ernsting, M. J.; Tang, W. L.; Maccallum, N.; Li, S. D., Synthetic modification of carboxymethylcellulose and use thereof to prepare a nanoparticle forming conjugate of docetaxel for enhanced cytotoxicity against cancer cells. Bioconjugate Chemistry 2011, 22 (12), 2474. DOI: 10.1021/bc200284b 11. Ernsting, M. J.; Tang, W. L.; Maccallum, N. W.; Li, S. D., Preclinical pharmacokinetic, biodistribution, and anti-cancer efficacy studies of a docetaxel-carboxymethylcellulose nanoparticle

in

mouse

models.

Biomaterials

2012,

33

(5),

1445-1454.

DOI:

10.1016/j.biomaterials.2011.10.061


21


Page 22 of 28

12. Ernsting, M. J.; Murakami, M.; Undzys, E.; Aman, A.; Press, B.; Li, S. D., A docetaxelcarboxymethylcellulose nanoparticle outperforms the approved taxane nanoformulation, Abraxane, in mouse tumor models with significant control of metastases. Journal of Controlled Release Official Journal of the Controlled Release Society 2012, 162 (3), 575-581. DOI: 10.1016/j.jconrel.2012.07.043 13. Lv, P. P.; Ma, Y. F.; Yu, R.; Yue, H.; Ni, D. Z.; Wei, W.; Ma, G. H., Targeted Delivery of Insoluble Cargo (Paclitaxel) by PEGylated Chitosan Nanoparticles Grafted with Arg-Gly-Asp (RGD). Molecular Pharmaceutics 2012, 9 (6), 1736-1747. DOI: 10.1021/mp300051h 14. Lv, P. P.; Wei, W.; Yue, H.; Yang, T. Y.; Wang, L. Y.; Ma, G. H., Porous quaternized chitosan nanoparticles containing paclitaxel nanocrystals improved therapeutic efficacy in nonsmall-cell lung cancer after oral administration. Biomacromolecules 2011, 12 (12), 4230-4239. DOI: 10.1021/bm2010774 15. Simi, C. K.; Emilia, A. T., Hydrophobic grafted and cross-linked starch nanoparticles for drug delivery. Bioprocess & Biosystems Engineering 2007, 30 (3), 173-180. DOI: 10.1007/s00449-007-0112-5 16. Mainka, D. I. H.; Laue, M.; Herrmann, D. I. A. S., Lignin Powder as a Filler for Thermoplastic Lightweight Design Components. Atz Worldwide 2015, 117 (3), 32-35. DOI: 10.1007/s38311-015-0171-1 17. Vilakati, G. D.; Hoek, E. M.; Mamba, B. B., Investigating the Usability of Alkali Lignin as an Additive in Polysulfone Ultrafiltration Membranes. Bioresources 2015, 10 (2), 3079-3096.


22

Page 23 of 28 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


18. Estellé, P.; Halelfadl, S.; Maré, T., Lignin as dispersant for water-based carbon nanotubes nanofluids: Impact on viscosity and thermal conductivity ☆. International Communications in Heat & Mass Transfer 2014, 57 (57), 8-12. DOI: 10.1016/j.icheatmasstransfer.2014.07.012 19. Lu, H.; Cornell, A.; Alvarado, F.; Behm, M.; Leijonmarck, S.; Li, J.; Tomani, P.; Lindbergh, G., Lignin as a Binder Material for Eco-Friendly Li-Ion Batteries. Materials 2016, 9 (3), 127. DOI:10.3390/ma9030127 20. Zhou, M.; Wang, W.; Yang, D.; Qiu, X., Preparation of a new lignin-based anionic/cationic surfactant and its solution behaviour. Rsc Advances 2014, 5 (4), 2441-2448. DOI: 10.1039/C4RA10524A 21. Hu, S. W.; Chen, S., A Multipurpose Lignin ‐ based Adsorbent for Metallic Ions, Nanoparticles and Various Organophosphate Pesticides in Hexane. Journal of the Chinese Chemical Society 2015, 62 (10), 875-888. DOI: 10.1002/jccs.201400299 22. Kai, D.; Tan, M. J.; Pei, L. C.; Yun, K. C.; Yong, L. Y.; Xian, J. L., Towards lignin-based functional materials in a sustainable world. Green Chemistry 2016, 18 (5), 1175-1200. DOI: 10.1039/C5GC02616D 23. Figueiredo, P.; Lintinen, K.; Kiriazis, A.; Hynninen, V.; Liu, Z.; Bauleth-Ramos, T.; Rahikkala, A.; Correia, A.; Kohout, T.; Sarmento, B., In vitro evaluation of biodegradable ligninbased nanoparticles for drug delivery and enhanced antiproliferation effect in cancer cells. Biomaterials 2017, 121, 97. DOI: 10.1016/j.biomaterials.2016.12.034


23


Page 24 of 28

24. Chen, N.; Dempere, L. A.; Tong, Z., Synthesis of pH-responsive Lignin Based Nanocapsules for Controlled Release of Hydrophobic Molecules. Acs Sustainable Chemistry & Engineering 2016, 4 (10). DOI: 10.1021/acssuschemeng.6b01209 25. Dai, L.; Liu, R.; Hu, L.; Zou, Z.; Si, C., Lignin nanoparticle as a novel green carrier for the efficient delivery of resveratrol. Acs Sustainable Chemistry & Engineering 2017, 5 (9). DOI: 10.1021/acssuschemeng.7b01903 26. Jiang, S.; Kai, D.; Dou, Q. Q.; Xian, J. L., Multi-arm carriers composed of antioxidant lignin core and poly(glycidyl methacrylate-co-poly(ethylene glycol) methacrylate) derivative arms for highly efficient gene delivery. Journal of Materials Chemistry B 2015, 3 (34), 68976904. DOI: 10.1039/C5TB01202C 27. Wang, M.; Hu, H.; Sun, Y.; Qiu, L.; Zhang, J.; Guan, G.; Zhao, X.; Qiao, M.; Cheng, L.; Cheng, L., A pH-sensitive gene delivery system based on folic acid-PEG-chitosan - PAMAMplasmid DNA complexes for cancer cell targeting. Biomaterials 2013, 34 (38), 10120-32. DOI: 10.1016/j.biomaterials.2013.09.006 28. Shen, Y.; Jin, E.; Zhang, B.; Murphy, C. J.; Sui, M.; Zhao, J.; Wang, J.; Tang, J.; Fan, M.; Kirk, E. V., Prodrugs Forming High Drug Loading Multifunctional Nanocapsules for Intracellular Cancer Drug Delivery. Journal of the American Chemical Society 2010, 132 (12), 4259. DOI: 10.1021/ja909475m 29. Dai, L.; Liu, K.; Si, C.; Wang, L.; Liu, J.; He, J.; Lei, J., Ginsenoside nanoparticle: A new green drug delivery system. Journal of Materials Chemistry B 2015, 4 (3), 529-538. DOI: 10.1039/C5TB02305J


24

Page 25 of 28 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


30. Dsouza, S. S.; Deluca, P. P., Development of a dialysis in vitro release method for biodegradable

microspheres.

Aaps

Pharmscitech

2005,

6

(2),

E323-E328.

DOI:

10.1208/pt060242 31. Zhang, G.; Yang, Z.; Lu, W.; Zhang, R.; Huang, Q.; Tian, M.; Li, L.; Liang, D.; Li, C., Influence of anchoring ligands and particle size on the colloidal stability and in vivo biodistribution of polyethylene glycol-coated gold nanoparticles in tumor-xenografted mice. Biomaterials 2009, 30 (10), 1928. DOI: 10.1016/j.biomaterials.2008.12.038 32. Fromm, M. F.; Kim, R. B.; Stein, C. M.; Wilkinson, G. R.; Dan, M. R., Inhibition of PGlycoprotein–Mediated Drug Transport A Unifying Mechanism to Explain the Interaction Between Digoxin and Quinidine. Circulation 1999, 99 (4), 552-7. 33. Oh, N.; Park, J. H., Endocytosis and exocytosis of nanoparticles in mammalian cells. International

Journal

of

Nanomedicine

2014,

2014

(Supplement

1),

51.

DOI:

10.2147/IJN.S26592 34. Iversen, T. G.; Skotland, T.; Sandvig, K., Endocytosis and intracellular transport of nanoparticles: Present knowledge and need for future studies. Nano Today 2011, 6 (2), 176-185. DOI: 10.1016/j.nantod.2011.02.003 35. Senior, J.; Delgado, C.; Fisher, D.; Tilcock, C.; Gregoriadis, G., Influence of surface hydrophilicity of liposomes on their interaction with plasma protein and clearance from the circulation: studies with poly(ethylene glycol)-coated vesicles. Biochimica Et Biophysica Acta 1991, 1062 (1), 77-82. DOI: 10.1016/0005-2736(91)90337-8


25


Page 26 of 28

36. Maeda, H.; Nakamura, H.; Fang, J., The EPR effect for macromolecular drug delivery to solid tumors: Improvement of tumor uptake, lowering of systemic toxicity, and distinct tumor imaging in vivo ☆ . Advanced Drug Delivery Reviews 2013, 65 (1), 71. DOI: 10.1016/j.addr.2012.10.002


26

Page 27 of 28 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


For Table of Contents Use Only

Development of novel lignin-based targeted polymeric nanoparticle platform for efficient delivery of anticancer drugs

Kefeng Liua, Dan Zhenga, Hantian Leia, Jing Liua, Jiandu Leia*, Luying Wanga*and Xingyuan Mab*


27


FA-PEG-AL/HCPT NPs were prepared via self-assembly for efficient delivery of anticancer drugs 99x54mm (300 x 300 DPI)


Page 28 of 28

Development of Novel Lignin-Based Targeted ... - ACS Publications

Recommend Documents