Fabrication and biological evaluation of chitosan coated hyaluronic acid-docetaxel conjugate nanoparticles in CD44+ cancer cells
© The Author(s). 2016
Received: 9 February 2016
Accepted: 21 July 2016
Published: 29 July 2016
Hyaluronic acid (HA) has been used for target-specific drug delivery because of strong affinity to CD44, a marker in which overexpressed in cancer cells and cancer stem cells. Conjugation of HA to the cytotoxic agents via active targeting can improve efficacy, biodistribution, and water solubility. To be able to benefit from passive targeting as well, a nanoparticulate system by counter ion using a polycation like chitosan may lead to a perfect delivery system.
Water soluble Hyaluronic acid-Docetaxel (HA-DTX) conjugate was prepared and used to formulate chitosan-coated HA-DTX nanoparticles by polyelectrolyte complex (PEC) method and optimized using Box-Behnken design. Biological evaluation of nanoparticles was done in CD44+ cancer cells.
Results and discussion
Biological evaluation of optimized formula showed IC50 of nanoparticles for 4 T1 and MCF-7 cell lines were 45.34 μM and 354.25 μM against 233.8 μM and 625.9 μM for DTX, respectively with increased cellular uptake showed by inverted confocal microscope.
Chitosan-coated HA-DTX nanoparticles were more effective against CD44+ cells than free DTX.
KeywordsGlyconanoparticles Nanomedicine Polyelectrolye Complex Macromolecular Drug Delivery Polysaccharides
Most of the anticancer drug products have systemic toxicity because of the wide uncontrolled distribution in the body. Besides, their lack of tumor localization and short half-lives are considerable obstacle facing effective cancer chemotherapy. Development of nanoparticulate drug delivery systems and polymer-drug conjugates of low molecular weight cytotoxic drug molecules to macromolecular carriers are effective ways to address these problems by enhanced permeation and retention (EPR) effect [1, 2]. In addition, conjugation of cytotoxic drugs to hydrophilic macromolecules can increase the water solubility of insoluble drugs such as docetaxel (DTX) and will enhance their biodistribution and therapeutic efficacy [3, 4].
DTX is an anticancer agent belongs to the Taxanes family and is a semi-synthetic derivative from the Taxus brevifolia  DTX shows its cytotoxic effect by inhibiting the depolymerization of microtubules and M-phase cell arrest . The conventional formulations of DTX in drug market suffering from low solubility of the active pharmaceutical ingredient which has been resolved using tween 80 as surfactant. This issue led to some complications in the clinic to control formulation-related adverse drug reactions . Due to the importance of this drug molecule in chemotherapy protocols, a lot of efforts dedicated to address novel formulations of DTX. In one of these approaches water-soluble macromolecular drug conjugates has been proposed to prepare tween-free formulations along with targeted drug delivery such as hyaluronic acid-docetaxel (HA-DTX) conjugates. In our previous report , although prepared conjugate had better solubility profile but the efficacy did not benefit from its polysaccharide CD44-targeting properties and heparine like effects of hyaluronic acid in blood circulation. Therefore this report is an attempt to add passive targeting enhanced permeation and retention of nanoparticulate system while keeping hyaluronate moiety. For this purpose, the water soluble conjugate became coated with chitosan by polyelectrolyte complex (PEC) method to prepare a nanoparticulate drug delivery system to improve pharmacokinetic features and efficacy.
For this purpose, hyaluronic acid (HA) as one of the common polysaccharides carriers has been used for conjugation of low molecular weight cytotoxic drugs such as DTX. HA is biocompatible, biodegradable and non-immunogenic . The most considerable advantage of HA is its strong affinity for CD44, a cell surface protein which is overexpressed in many cancer cells and cancer stem cells [9, 10]. CD44 is a specific biological receptor for HA [11, 12]. HA could have an enhanced attachment and uptake into malignant cells with metastatic activities  and has been used for target-specific drug delivery [14, 15]. We hypothesized using HA as carrier and targeting moiety simultaneously may reverse the multiple drug resistance of cancer stem cells via affecting CD44 and regarding physical correlation of P-gp and these markers . In this regard, HA-DTX conjugates has been prepared and evaluated which showed suitable efficacy and safety profile. This research reports optimized polyelectrolyte complex nanoparticles using HA-DTX as cationic part.
Polyelectrolyte complexes which were prepared by electrostatic interaction between unlike charged poly-ions have received substantial attention in drug delivery systems. The synthesis of PEC nanoparticles is simple and can be easily carried out under mild conditions without using toxic organic solvents or chemical cross-linkers . In the present study, chitosan as a N-deacetylated derivative of chitin has been used extensively as a biocompatible polysaccharide  with a cationic nature that can be protonated in weak acidic environment , and therefore it can improve the bioavailability of DTX .
Achieving optimization of the nanoparticle preparation could be performed by classical method of changing one variable at a time while others have been remained constant. However this method needs lots of series of experiments and time. Moreover in this approach the possible interaction between independent factors will not be observed. Therefore, the fine optimized formulation will not be achieved. Design-of-experiment (DoE) method has been used in pharmaceutical studies to solve this problem. Optimization by response surface methodology including Box-Behnken method is considered as the major application of DoE [21, 22].
In the present study, the water soluble conjugated HA-DTX was synthesized. Then, chitosan coated HA-DTX nanoparticles were prepared by PEC method considering the anionic structure of HA-DTX and cationic chitosan. Box-Behnken statistical design using a response surface methodology has been employed to obtain the optimized condition in terms of particle size, size distribution, drug loading and zeta potential. For determination of efficacy of optimized chitosan coated HA-DTX conjugate nanoparticles, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed on MCF-7, human cancer cell line, and 4 T1 mouse breast cancer cell line. MCF-7 and 4 T1 cell lines were also used for cell uptake study.
Materials and methods
Sodium hyaluronate (MW 25 kDa) was purchased from GuangLong (Shandong, China). Anhydrous DTX was purchased from Jiangsu Yew (Jiangsu, China). Chitosan (MW 50 kDa, (Primex, Karmoy, Norway), 1-Ethyl–3-[3-(dimethylamino)-propyl] carbodiimide (EDC), N-hydroxy succinimide (NHS), 4’,6-diamidino-2-phenylindole (DAPI) and triethylamine were purchased from Sigma Aldrich (Seelze, Germany). MTT dye was from Merck (Darmstadt, Germany). Dulbecco's Modified Eagle Medium (DMEM) with high glucose, RPMI 1640, FBS (Fetal Bovine Serum), trypsin, penicillin and streptomycin were purchased from Biosera (Vienna, Austria). Ultra-purified water was used throughout the analysis and all other chemicals were of analytical grade. 4 T1 and MCF-7 cell lines were obtained from National Cell Bank of Iran (Pasteur Institute of Iran, Tehran, Iran).
Synthesis of hyaluronic acid-docetaxel (HA-DTX) conjugates
At first for preparing desalted HA, HA (MW 25 kDa) (1 g) was dissolved in 100 mL of deionized water, then the solution was dialyzed (MWCO 12 kDa) in deionized water for 24 h and lyophilized .
Desalted-HA (400 mg) was dissolved in 80 mL of deionized water. Then EDC and NHS were added to the solution in 11 and 10 molar ratios of carboxyl groups of HA, respectively. The mixture was stirred at 40 °C for 3 h. Then 80 mL dimethylformamide (DMF) containing 400 mg of DTX and 20 mL of triethylamine were added. After 24 h the mixture was refluxed at 70 °C and after cooling to room temperature was transferred to pretreat dialysis tubing (MWCO 3500 kDa). The mixture was purified by dialysis against water-acetone solution (50:50, v/v) for 1 h, water-acetone (75:25, v/v) for 1 h and water for 1 h, respectively. The obtained HA-DTX conjugate is water soluble. To eliminate the remained DTX, the mixture was transferred to a separating funnel and extracted three times with dichloromethane. The aqueous phase was lyophilized. Chemical integrity of the resulted product was checked by 1H-NMR (Bruker AC 500 Spectrophotometer, Germany) and fourier transform infra-red (FTIR) spectroscopy (Nicolet Magna-FTIR 550 Spectrometer, WI, USA). The concentration of DTX was measured by UV Spectrophotometry (UV-visible Spectrophotometer, 160A, SHIMADZU, Japan) at 229 nm versus a suitable blank solution containing the appropriate concentration of HA.
Preparation of chitosan coated HA-DTX conjugate nanoparticle
Chitosan coated HA-DTX conjugate nanoparticles were prepared by PEC method, considering the anionic structure of HA-DTX conjugates and cationic chitosan . As a representative example, 1 mL of aqueous solution of HA-DTX (4.25 mg/mL) was added slowly to 1 mL of dilute chitosan solution (0.250 mg/mL of 1 % acetic acid) in 1 min while stirring at 370 rpm at room temperature. It should be mentioned that during the screening and optimization procedure, various factors including ratios of HA-DTX conjugate to chitosan, stirring rate and temperature were evaluated.
DTX determination in the conjugates and nanoparticles
Fourier transform infra-red spectroscopy of conjugates
Freeze-dried conjugated HA-DTX, HA, DTX were analyzed by FTIR Spectrometer. The data was achieved in the range of 400–4000 cm−1 for each sample. The FTIR spectra of conjugated HA-DTX were compared with pure substances.
Experimental design studies
Variables used in Box-Behnken experimental design
Particle size (nm)
Stirring rate (rpm)
Zeta potential (mv)
Ratio of HA-DTX conjugate to chitosan
Polydispersity index (PdI)
Drug content or DTX loading (%)
The aim of the design was to achieve to the optimum formulation, with both minimum size and maximum loading. The PdI factor should be at possible lowest level and zeta potential should be appropriate to have stable nanoparticles. Obtained responses from three optimized formulations were compared with the suggested experimental responses to evaluate the precision of model.
Characterization of the nanoparticles
The size and zeta potential of the nanoparticles were determined using a Zetasizer Nano ZS Analyzer (Malvern Instruments, UK) with a He-Ne laser beam at wavelength of 633 nm at 25 °C.
Surface morphology of the nanoparticles observed using Scanning Electron Microscopy (SEM) (Philips Xl30, The Netherlands) and Atomic Force Microscopy (AFM) (dualscope™ DS 95-200/50, Denmark) microscopy. For AFM evaluation, one drop of nanoparticle suspension was dried on the surface of clean silicon wafer at room temperature. AFM study was performed with 20 μm scanner in tapping mode. For SEM imaging, dried nanoparticles were gently coated by gold layer with a sputter coater and evaluated at 30 kV using a 6300 field emission scanning electron microscope.
Differential scanning calorimetry (DSC) was performed using Mettler-Toledo DSC822e (Greifensec, Switzerland) and data acquisition and analysis was carried out by a software package of STARe 9.01. The system was conducted by using 8 mg of sample, deposited in 40 μL aluminum pans and hermetically sealed, under a nitrogen gas dynamic flow at a scanning heating rate of 10 °C/min over a range of 20 °C to 300 °C. Empty hermetically sealed aluminum pan was used as a control.
Cytotoxicity evaluation of nanoparticles
Preparation of fluorescent-labeled HA
To prepare fluorescent-labeled HA conjugate, HA was labeled with fluorescamine. One hundred mg of HA was dissolved in 10 mL water and 61.5 mg of EDC and 45.5 mg of NHS were added. The mixture was stirred for 3 h and then 45.84 mg fluorescamine was added and stirred for 24 h. The reaction flask was protected from light by covering with an aluminum foil. After 24 h the mixture was purified by dialysis (MWCO 3500) against deionized water and finally the product was freeze-dried. Fluorescent-labeled nanoparticles were also prepared by fluorescent-labeled HA conjugate instead of HA-DTX conjugate and the nanoparticle preparation method was the same as the optimized method used for normal nanoparticles.
Cell uptake studies
In order to study the cellular uptake of the nanoparticles, 4 T1 and MCF-7 cell lines were seeded at 1 × 105 cell/well in a cover glass and incubated at 37 °C with 5 % CO2 atmosphere for 24 h. After complete adhesion, the medium was carefully removed and replaced with fresh medium containing fluorescent-labeled nanoparticles and incubated for 2 h. In this stage, the medium containing drug was removed and cells were washed 4 times with PBS and fixed with formaldehyde 4 % for 4 min. Nuclear coloring was performed with DAPI (0.5 mg/mL) in 5 min and then cells were washed 4 times with PBS using inverted confocal microscope (Nikon ECLIPSE Ti, Tokyo, Japan) cell images were taken .
SPSS 20.0 statistical software and one-way analysis of variance (ANOVA) were used to assess the data groups. All the results were evaluated as mean ± standard deviation (SD). Significance difference of p < 0.05 was accepted.
Results and discussion
Synthesis and characterization of water-soluble HA-DTX direct conjugate
The absorption of DTX in conjugate was measured by UV spectroscopy. According to UV absorption, it was shown that 1 mg of HA-DTX conjugate contains 69 μg of DTX.
Preparation of chitosan coated HA-DTX conjugate nanoparticle
Publications reported several nanoparticular based methods to improve DTX drug delivery to cancer cells (DTX loaded chitosan nanoparticle , targeted DTX nanoparticles with different targeting agents like folic acid  and albumin nanoparticles of DTX ). HA could cause heparin-induced thrombocytopenia because of its nature as a polysaccharide . This event should be considered if the HA-DTX used alone. Coating the HA-DTX conjugate with chitosan may limit the exposure of HA with platelet in blood circulation and reduce thrombocytopenia. Chitosan-coated HA-DTX conjugate nanoparticles were prepared based on these findings.
The effect of independent variables on dependent variables
Stirring rate (rpm)
Ratio of HA-DTX conjugate to chitosan
Particle size (nm)
Zeta potential (mv)
Polydispersity index (PdI)
DTX loading (%)
which A, B and C are independent variables, and Y is the predicted dependent factor, b0 is the intercept, b1, b2, and b3 are linear coefficients, b11, b22, and b33 are squared coefficients, and b12, b13, and b23 are the interaction coefficients of equation.
Size of nanoparticles
Confirmation of designed optimized experiments
Analysis of variance (ANOVA) and lack of fit parameters for the responses according to quadratic model
Sum of squares
Degrees of freedom (df)
Quadratic vs 2FI
Quadratic vs 2FI
Quadratic vs 2FI
Quadratic vs 2FI
Lack of Fit
Three experiments were performed in the lab according to the optimized formulation and there was no significant difference between the obtained and predicted results. The mean amount obtained in these experiments was 234 nm for size of nanoparticles, 0.088 for PdI and 3.18 % for DTX content and +20.03 mV for zeta potential. The mean entrapment efficiency for these experiments was 62.78 %. The entrapment efficiency showed that nanoparticles could be an effective carrier for DTX.
Characterization of nanoparticles
Particle size,distribution and zeta potential
Differential scanning calorimetry
Freeze-dried nanoparticles, conjugated HA-DTX, chitosan, HA, DTX and physical mixture of HA-DTX conjugate and chitosan was analyzed during predetermined increasing temperature rate to obtain DSC thermogram.
DSC thermogram specificities of HA has an influence on DSC thermogram of physical mixture of HA-DTX conjugate and chitosan. Because of the ionic charge of the chitosan (cationic) and HA (anionic), these two compounds can form a charge-transfer bond (the same as PEC formation) which may attenuate the difference in comparing these physical mixture and polyelectrolyte complex. So, DSC thermogram of nanoparticles may not express this influence. These results revealed that chitosan coated nanoparticles protected conjugated HA-DTX and weakened the effect of temperature, which showed the formation of chitosan coated HA-DTX nanoparticles.
Chitosan coated HA-DTX nanoparticles were morphologically studied by SEM and AFM. SEM showed (Fig. 5c) that nanoparticles are uniform spheres and non-aggregated. AFM micrographs (Fig. 5d) confirmed the spherical shape of nanoparticles too.
In vitro cytotoxicity
Cell uptake studies
Entrance of nanoparticles in cancer cells had a direct relation with their observed cytotoxic effect. Free DTX molecules could be transported out by P-glycoprotein (P-gp) pumps, but drug loaded nanoparticles were taken up by cells through an endocytosis pathway. The result represented higher cellular uptake of nanoparticles because of their ability to escape from the effect of P-gp pumps . The uptake of optimized FITC-labeled chitosan-coated HA-DTX conjugate nanoparticles by 4 T1 and MCF-7 after 24 h incubation is shown in Fig. 7c. No treatment cells of each cell line are presented in Fig. 7c as a control. Based on fluorescence intensity, FITC-labeled chitosan-coated HA-DTX nanoparticles showed appropriate entrance into 4 T1 and MCF-7 cells. As a result it can be proposed that developed nanoparticles could bring loaded drug molecules effectively to cell cytoplasm as a novel drug delivery system.
Water soluble HA-DTX conjugate was prepared according to HA strong affinity for CD44, a cell surface protein which is overexpressed in many cancer cells and cancer stem cells. Chitosan-coated HA-DTX nanoparticles by polyelectrolyte complex method improved DTX availability. The fine optimized formulation was achieved with proper particle size, PdI, zeta potential and drug loading. The biological evaluation of nanoparticles showed they were more potent than free DTX in cytotoxic effect on MCF-7 and 4 T1 cells beside of their appropriate entrance in to cells. These findings need further evaluation to take into account potential improved pharmacokinetic of nanoparticulate drug delivery system.
NSR carried out all experiments and drafted the manuscript. NG supervised and finalized the paper. FA participated in nanoparticles fabrication. MA conducted structure elucidation. EF revised the manuscript and helped in analysis. MRK lead statistical analysis and DOE. ZHM helped in cellular studies. FA supervised all pharmaceutical and cellular studies. RD supervised all experiments and approved the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Goodarzi N, Varshochian R, Kamalinia G, Atyabi F, Dinarvand R. A review of polysaccharide cytotoxic drug conjugates for cancer therapy. Carbohydr Polym. 2013;92:1280–93.View ArticlePubMedGoogle Scholar
- Tavassolian F, Kamalinia G, Rouhani H, Amini M, Ostad SN, Khoshayand MR, Atyabi F, Tehrani MR, Dinarvand R. Targeted poly (l-γ-glutamyl glutamine) nanoparticles of docetaxel against folate over-expressed breast cancer cells. Int J Pharm. 2014;467:123–38.View ArticlePubMedGoogle Scholar
- Dosio F, Stella B, Arpicco S, Cattel L. Macromolecules as taxane delivery systems. Expert Opin Drug Deliv. 2011;8:33–55.View ArticlePubMedGoogle Scholar
- Goodarzi N, Ghahremani MH, Amini M, Atyabi F, Ostad SN, Shabani Ravari N, Nateghian N, Dinarvand R. CD44-targeted docetaxel conjugate for cancer cells and cancer stem-like cells: A novel hyaluronic acid-based drug delivery system. Chem Biol Drug Des. 2014;83:741–52.View ArticlePubMedGoogle Scholar
- Mugabe C, Liggins RT, Guan D, Manisali I, Chafeeva I, Brooks DE, Heller M, Jackson JK, Burt HM. Development and in vitro characterization of paclitaxel and docetaxel loaded into hydrophobically derivatized hyperbranched polyglycerols. Int J Pharm. 2011;404:238–49.View ArticlePubMedGoogle Scholar
- Akhlaghi SP, Saremi S, Ostad SN, Dinarvand R, Atyabi F. Discriminated effects of thiolated chitosan-coated pMMA paclitaxel-loaded nanoparticles on different normal and cancer cell lines. Nanomedicine. 2010;6:689–97.PubMedGoogle Scholar
- Cho HJ, Yoon HY, Koo H, Ko SH, Shim JS, Lee JH, Kim K, Chan Kwon I, Kim DD. Self-assembled nanoparticles based on hyaluronic acid-ceramide (HA-CE) and Pluronic ® for tumor-targeted delivery of docetaxel. Biomaterials. 2011;32:7181–90.View ArticlePubMedGoogle Scholar
- Menzel EJ, Farr C. Hyaluronidase and its substrate hyaluronan: Biochemistry, biological activities and therapeutic uses. Cancer Lett. 1998;131:3–11.View ArticlePubMedGoogle Scholar
- Dalerba P, Cho RW, Clarke MF. Cancer stem cells: models and concepts. Annu Rev Med. 2007;58:267–84.View ArticlePubMedGoogle Scholar
- Aruffo A, Stamenkovic I, Melnick M, Underhill CB, Seed B. CD44 is the principal cell surface receptor for hyaluronate. Cell. 1990;61:1303–13.View ArticlePubMedGoogle Scholar
- Auzenne E, Ghosh SC, Khodadadian M, Rivera B, Farquhar D, Price RE, Ravoori M, Kundra V, Freedman RS, Klostergaard J. Hyaluronic acid-paclitaxel: antitumor efficacy against CD44 (+) human ovarian carcinoma xenografts. Neoplasia. 2007;9:479–86.View ArticlePubMedPubMed CentralGoogle Scholar
- Stamenkovic I, Aruffo A, Amiot M, Seed B. The hematopoietic and epithelial forms of CD44 are distinct polypeptides with different adhesion potentials for hyaluronate-bearing cells. EMBO J. 1991;10:343.PubMedPubMed CentralGoogle Scholar
- Herrlich P, Sleeman J, Wainwright D, König H, Sherman L, Hilberg F, Ponta H. How tumor cells make use of CD44. Cell Commun Adhes. 1998;6:141–7.View ArticleGoogle Scholar
- Kurisawa M, Chung JE, Yang YY, Gao SJ, Uyama H. Injectable biodegradable hydrogels composed of hyaluronic acid–tyramine conjugates for drug delivery and tissue engineering. Chem Commun (Camb). 2005;14:4312–4.View ArticleGoogle Scholar
- Lee H, Mok H, Lee S, Oh Y-K, Park TG. Target-specific intracellular delivery of siRNA using degradable hyaluronic acid nanogels. J Control Release. 2007;119:245–52.View ArticlePubMedGoogle Scholar
- Misra S, Ghatak S, Toole BP. Regulation of MDR1 expression and drug resistance by a positive feedback loop involving hyaluronan, phosphoinositide 3-kinase, and ErbB2. J Biol Chem. 2005;280:20310–5.View ArticlePubMedGoogle Scholar
- Polexe RC, Delair T. Elaboration of stable and antibody functionalized positively charged colloids by polyelectrolyte complexation between chitosan and hyaluronic acid. Molecules. 2013;18:8563–78.View ArticlePubMedGoogle Scholar
- Park JH, Kwon S, Lee M, Chung H, Kim J-H, Kim Y-S, Park R-W, Kim I-S, Seo SB, Kwon IC. Self-assembled nanoparticles based on glycol chitosan bearing hydrophobic moieties as carriers for doxorubicin: in vivo biodistribution and anti-tumor activity. Biomaterials. 2006;27:119–26.View ArticlePubMedGoogle Scholar
- Kim SJ, Yoon SG, Lee KB, Park YD, Kim SI. Electrical sensitive behavior of a polyelectrolyte complex composed of chitosan/hyaluronic acid. Solid State Ion. 2003;164:199–204.View ArticleGoogle Scholar
- Saremi S, Dinarvand R, Kebriaeezadeh A, Ostad SN, Atyabi F. Enhanced oral delivery of docetaxel using thiolated chitosan nanoparticles: preparation, in vitro and in vivo studies. Biomed Res Int. 2013;2013:150478.View ArticlePubMedPubMed CentralGoogle Scholar
- Jafari Malek S, Khoshchehreh R, Goodarzi N, Khoshayand MR, Amini M, Atyabi F, Esfandyari-manesh M, Tehrani S, Mohammad Jafari R, Maghazei MS, et al. Cis-Dichlorodiamminoplatinum (II) glyconanoparticles by drug-induced ionic gelation technique targeted to prostate cancer: Preparation, optimization and in vitro characterization. Colloids Surf B Biointerfaces. 2014;122:350–8.View ArticlePubMedGoogle Scholar
- Azadi A, Hamidi M, Khoshayand M-R, Amini M, Rouini M-R. Preparation and optimization of surface-treated methotrexate-loaded nanogels intended for brain delivery. Carbohydr Polym. 2012;90:462–71.View ArticlePubMedGoogle Scholar
- Lee H, Lee K, Park TG. Hyaluronic acid − paclitaxel conjugate micelles: Synthesis, characterization, and antitumor activity. Bioconjug Chem. 2008;19:1319–25.View ArticlePubMedGoogle Scholar
- Denuziere A, Ferrier D, Damour O, Domard A. Chitosan–chondroitin sulfate and chitosan–hyaluronate polyelectrolyte complexes: biological properties. Biomaterials. 1998;19:1275–85.View ArticlePubMedGoogle Scholar
- Luo Y, Prestwich GD. Synthesis and selective cytotoxicity of a hyaluronic acid-antitumor bioconjugate. Bioconjug Chem. 1999;10:755–63.View ArticlePubMedGoogle Scholar
- Manoochehri S, Darvishi B, Kamalinia G, Amini M, Fallah M, Ostad SN, Atyabi F, Dinarvand R. Surface modification of PLGA nanoparticles via human serum albumin conjugation for controlled delivery of docetaxel. Daru. 2013;21:58.View ArticlePubMedPubMed CentralGoogle Scholar
- Lee JY, Choi YS, Suh JS, Kwon YM, Yang VC, Lee SJ, Chung CP, Park YJ. Cell‐penetrating chitosan/doxorubicin/TAT conjugates for efficient cancer therapy. Int J Cancer. 2011;128:2470–80.View ArticlePubMedGoogle Scholar
- Nateghian N, Goodarzi N, Amini M, Atyabi F, Khorramizadeh MR, Dinarvand R. Biotin/folate‐decorated human serum albumin nanoparticles of docetaxel: comparison of chemically conjugated nanostructures and physically loaded nanoparticles for targeting of breast cancer. Chem Biol Drug Des. 2015;87(1):69–82.View ArticlePubMedGoogle Scholar
- Li C, Yu D-F, Newman RA, Cabral F, Stephens LC, Hunter N, Milas L, Wallace S. Complete regression of well-established tumors using a novel water-soluble poly (L-glutamic acid)-paclitaxel conjugate. Cancer Res. 1998;58:2404–9.PubMedGoogle Scholar
- Jain A, Thakur K, Kush P, Jain UK. Docetaxel loaded chitosan nanoparticles: Formulation, characterization and cytotoxicity studies. Int J Biol Macromol. 2014;69:546–53.View ArticlePubMedGoogle Scholar
- Gandhi NS, Mancera RL. The structure of glycosaminoglycans and their interactions with proteins. Chem Biol Drug Des. 2008;72:455–82.View ArticlePubMedGoogle Scholar
- Saremi S, Atyabi F, Akhlaghi SP, Ostad SN, Dinarvand R. Thiolated chitosan nanoparticles for enhancing oral absorption of docetaxel: Preparation, in vitro and ex vivo evaluation. Int J Nanomedicine. 2011;6:119–28.PubMedPubMed CentralGoogle Scholar
- Panyam J, Labhasetwar V. Dynamics of endocytosis and exocytosis of poly (D, L-lactide-co-glycolide) nanoparticles in vascular smooth muscle cells. Pharm Res. 2003;20:212–20.View ArticlePubMedGoogle Scholar