Biomaterials 35 (2014) 5171e5187

Contents lists available at ScienceDirect

Biomaterials journal homepage: www.elsevier.com/locate/biomaterials

Doxorubicin-loaded, charge reversible, folate modified HPMA copolymer conjugates for active cancer cell targeting Lian Li, Qingqing Yang, Zhou Zhou, Jiaju Zhong, Yuan Huang* Key Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education, West China School of Pharmacy, Sichuan University, No. 17, Block 3, Southern Renmin Road, Chengdu 610041, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 9 January 2014 Accepted 12 March 2014 Available online 1 April 2014

Although folate exhibits many advantages over other targeting ligands, it has one major defect: poor water solubility. Once it was conjugated to hydrophilic drug carrier such as N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer, the hydrophobic folate may be buried inside the random polymer coil and not exposed to be accessible to its receptor on the cell surface, thus losing its active targeting ability. To address this folate dilemma, the positive charge was introduced in the present study. The obtained cationic folate-functionalized HPMA copolymers exhibited a synergistic enhancing effect on cellular uptake by folate receptor (FR) positive Hela cells via electrostatic absorptive endocytosis and folate receptor-mediated endocytosis, with the involvement of multiple internalization pathways including clathrin-mediated endocytosis, caveolae-mediated endocytosis, macropinocytosis and energydependent endocytosis. As demonstrated in binding efficiency study, the FR antibody bound to 71.2% of tested cells in the competition with neutral folate modified HPMA copolymers, while the FR antibodybounded cells decreased to only 34.0% in competition with cationic folate modified HPMA copolymers, indicating that the positively charge could probably amplify the binding efficiency of folate to its receptor due to close proximity of the conjugates to the cell surface by the electronic adhesion. In addition, the cell uptake study on FR negative A549 cells also confirmed the specific role of folate as targeting ligand. Then, to avoid non-specific binding by positive charge in the circulation, the charge shielding/deshielding approach was further employed. With selective hydrolysis of the charge shielding groups 2,3dimethylmaleic anhydride (DMA) at tumor extracellular pH 6.8, the conjugates underwent a quick charge-reversible process with more than 80% DMA cleavage within 2 h and endocytosed into the endo/ lysosomes much more rapidly than at physiological pH 7.4. And then the drug release was triggered by the cleavage of hydrazone spacer at another level of pH 5 in endo/lysosomal compartment. Furthermore, the anticancer activity results showed that Dox-loaded, charge-switchable, folate modified HPMA copolymer conjugates could indeed lead to enhanced cytotoxicity, stronger apoptosis and greater tumor spheroid inhibition towards Hela cells, indicating the great potential feasibility of this multiple responsive drug delivery system. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: HPMA copolymer Charge reverse Tumor extracellular acidity Folate dilemma

1. Introduction Among the variety of polymers frequently applied as drug carriers for cancer therapy, water soluble N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers emerged as one of the most promising candidates owing to their unique characteristics such as good compatibility, non-immunogenicity, non-toxicity and multifunctionality for the easy conjugation of various drugs and targeting moieties [1e3]. Small molecular drugs covalently bound to

* Corresponding author. Tel./fax: þ86 28 85501617. E-mail address: [email protected] (Y. Huang). http://dx.doi.org/10.1016/j.biomaterials.2014.03.027 0142-9612/Ó 2014 Elsevier Ltd. All rights reserved.

HPMA copolymers have shown superior anticancer efficiency over the original ones due to the improved pharmacokinetics and biodistribution profiles via the enhanced permeability and retention (EPR) effect [4,5]. However, the poor cell membrane affinity of HPMA polymers, inherited from their highly hydrophilic and neutral electric physiochemical properties, appear to negatively affect their cellular internalization. The EPR effect for the passive accumulation in the tumor tissues might not always be sufficient enough, while effective delivery of drugs to the tumor cells or subcellular organelles seems to be a more important issue [6e8]. The most commonly used alternative is to add targeting ligands that specifically bind to the receptors over-expressed on the cell surface [9e11]. Folate, which is extensively conjugated to different

5172

L. Li et al. / Biomaterials 35 (2014) 5171e5187

drug carriers, has demonstrated extremely successful delivery to numerous folate-receptor positive tumor cells both in vitro and in vivo due to its high binding affinity (Kd z 1010 M) and specificity, small size, convenient modification, commercially availability and low cost [12,13]. However, despite all those promising merits, folate with relatively poor water solubility was reported to show lower level of exposure and accessibility to folate-receptor on the cell surface [14e16]. The conformation change was demonstrated to occur when incorporating the folate residue (5 mol%) into the HPMA copolymers, which led to barely enhanced drug uptake by the folate-receptor positive cells. The folate was presumably hidden in the hydrophilic corona of the random polymer coil thus hampers the ligandereceptor interaction [14]. Similar results that the hydrophobicity of folate clearly affected its exposure were also found in folate targeted PEG-doxorubicin polymer [15] and PLGA-PEGFolate nanoparticle [16]. Although increasing the folate content to high levels may lead to saturated folate concentration in the hydrophobic interior of hydrophilic polymer chain and could increase the amount of folate presented on the surface [14], this may also consequently reduce the water solubility of the carrier, negatively influence drug release profile and undesirably increase the phagocytosis by macrophages in the circulation [17,18]. Herein, all those serious issues regarding the loss of folate targeting activity in anticancer drug delivery are referred to as the ‘folate dilemma’. Therefore, a rational strategy designed to make the most use of folate as targeting ligands is required. In the current study, in order to maximally exploit the targeting efficiency of folate and further enhance the cell uptake of folate modified HPMA-doxorubicin conjugates, cationic charge was introduced. On one hand, cationic carriers have been well-known to undergo a quick binding process onto the negatively charged cell membranes, followed by rapid drug uptake by the cell, which was dominant over molecular weight or hydrophobic property in the effect on cellular uptake of HPMA copolymers [19]. On the other hand, we hypothesized that upon electronic cell membrane adhesion which brings the conjugate in close proximity to the cell surface, the chance for the folate to interact with the folate-receptor might also increase, leading to a synergic enhancing effect on cell uptake by both ligandereceptor interaction and electronic attraction. Unfortunately, the positive charge, if not masked before reaching the tumor, could also cause non-specific binding to all the cells encountered in the circulation [20]. This problem could be avoided by employing cationic charge shielding/deshielding approach [21e 25]. As tumor tissues have a more acidic extracellular environment (pH w 6.8) than normal physiology pH 7.4, the anionic 2,3-dimethylmaleic anhydride (DMA) as the shielding group could be stably attached to the positively charged primary amine in the circulation but can be cleaved to regenerate the positive charge immediately in response to the mild acidity of tumor tissues [26e28]. It’s convincible that after the arrival at tumor site via EPR effect, by utilizing a local stimuli of the tumor extracellular acidity to trigger charge conversion, an additional selectivity and accumulation would be gained [29,30]. Herein, in the present study, the folate-decorated, chargeswitchable, doxorubicin-loaded conjugates based on the HPMA copolymers are designed. The cellular uptake, anticancer activity and relative mechanisms of the dual-modified conjugates were investigated and compared with that of the single modification used separately. 2. Materials and methods 2.1. Materials Doxorubicin hydrochloride (DOX$HCl) was purchased from Huafeng United Technology Co., Ltd. (Beijing, China). Fluorescein isothiocyanate (FITC) and 2,3-

dimethylmaleic anhydride (DMA) were obtained from Acros organics. Folic acid, 2,4,6-Trinitrobenzene-1-sulfonic acid (TNBSA), fluorescamine, 3-(4, 5-dimethyl-2tetrazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) and 40 , 6-diamidino-2phenylindole (DAPI) were purchased from SigmaeAldrich (St. Louis, MO, USA). 1[3-(Dimethylamino) propyl]-3-ethylaarbodiimide hydrochloride (EDC.HCl) was gained from Meapeo Co., Ltd. (Shanghai, China). N-3-aminopropylmethacrylamide hydrochloride (APMA) was purchased from PolySciences. Lysotracker Green was purchased from Invitrogen (Carlsbad, CA). The primary antibody MAb MOv18 and secondary antibody FITC-conjugated goat anti-rabbit IgG(H þ L) were purchased from ABclonal. 2.2. Synthesis and characterization of various conjugates based on HPMA copolymers 2.2.1. Synthesis of monomers The monomers of N-(2-hydroxypropyl) methacrylamide (HPMA), N-methacryloyl-aminopropyl-fluorescein-5-isothiocyanate (MA-AP-FITC) and N-methacryloylglycylglycyl-hydrazide-doxorubicin (Ma-GG-NHN ¼ Dox) were synthesized according to previous reports [31,32]. 2.2.2. Synthesis of polymeric conjugates HPMA copolymer-doxorubicin conjugates (HPMA-Dox, polymer 13) were prepared by radical solution polymerization in methanol (AIBN, 2 wt.%; monomer concentration 12.5 wt.%; molar ratio HPMA/MA-GG-NHN ¼ Dox was 92.5:7.5). The copolymerization was carried out in sealed ampoules under nitrogen at 50  C for 24 h. The copolymer was isolated from polymerization mixture by precipitated into diethyl ether, and then purified by dialysis against distilled water for one day and freeze-dried. The folate-decorated HPMA copolymer-Doxorubicin conjugates (FA-HPMA-Dox, polymer 14) were synthesized by two steps. Firstly, the polymer precursor was prepared by random radical solution copolymerization in methanol (AIBN, 2 wt.%; monomer concentration 12.5 wt.%; molar ratio HPMA/APMA/MA-GG-NHN ¼ Dox was 87.5:5:7.5). Secondly, the folic acid with an equal molar amount of APMA monomer was dissolved in DMSO, and then 14 equivalents of EDC.HCl was added and stirred for 1 h at room temperature. The polymer precursor was dissolved in dimethyl sulfoxide and then a desired amount of triethylamine was added to adjust the solution to pH 8.5. The activated folate and polymer precursor solutions were mixed and stirred for 2 days, followed by purification by dialysis against distilled water for 2 days. The resultant solution was finally freeze-dried to obtain FA-HPMADox (polymer 14). The doxorubicin-loaded, folate-decorated, DMA-protected HPMA copolymer (FA-APMA-DMA-Dox, polymer 16) was synthesized as illuminated in Fig. 1. First, positively charged amine groups containing polymer precursor (APMA-Dox) was prepared by radical solution copolymerization in methanol (AIBN, 2 wt.%; monomer concentration 12.5 wt.%; molar ratio HPMA/APMA/MA-GG-NHN ¼ Dox was 72.5:20:7.5). Then APMA-Dox was reacted with 1/4 equivalent (to APMA monomer) of free folate to obtain the positive charged folate-modified copolymer FA-APMADox. Finally, APMA-Dox or FA-APMA-Dox was dissolved in sodium bicarbonate buffer (0.1 M, pH 8.5), and 3 equivalents (to APMA monomer) of 2,3-dimethylmaleic anhydride (DMA) was slowly added. With the addition of DMA, the pH decreased rapidly and was maintained in the range of 8.0e8.5 by simultaneous addition of 0.2 N NaOH. After all DMA was added, the solution was stirred for 4 h and then was dialyzed against sodium bicarbonate buffer (0.1 M, pH 8.5) for 2 days followed by lyophilization to obtain the DMA-protected conjugates APMA-DMA-Dox (polymer 15) or FA-APMA-DMA-Dox (polymer 16). For different purposes, the corresponding non-drug loaded conjugates (polymers 1e6) or FITC-labeled conjugates (polymers 7e12) were synthesized by the similar methods, except that the MA-GG-NHN ¼ Dox monomer was absent or replaced with MA-AP-FITC (2 mol% in feed). To synthesize the Dox-loaded HPMA copolymers containing different folate contents, the polymer precursors containing different amount of APMA monomer was prepared by radical solution copolymerization as described above with the feeding molar ratio of APMA monomer being 5%, 10% and 15%. Then the three precursors were reacted with the free folic acid with an equal molar amount of APMA monomer as described to obtain the polymers 17e19. 2.3. Characterization of various conjugates based on HPMA copolymers The molecular weight and poly dispersity index (PDI) of the conjugates were estimated by size exclusion chromatography on a Superose 200 10/300GL analytical column (Amersham Biosciences, NJ) calibrated with poly (HPMA) fractions using a Fast Protein Liquid Chromatography (AKTA FPLC) system (Amersham Biosciences, NJ). Similar to the method described previously, UVeVis spectroscopy was used to quantify the amount of folate [33], FITC [19] and Dox [34] conjugated onto the copolymers. Briefly, folate modified copolymers were dissolved in 0.1 M borate buffer (pH 9.0) and the absorbance of the solutions at 280 nm was measured. Using a calibration curve obtained by measuring the absorbance of different concentrations of free folate in 0.1 M borate buffer (pH 9.0) at 280 nm and an average molar absorption coefficients of 20,650 M1 cm1, the folate content in the copolymer was calculated. Similarly, the content of FITC or Dox were also determined by UVeVis

* O

O

O

O NH

AIBN

O NH

50 °C OH

NH

NH O

C

OH

H

OO

OO OH NH 2

OH NH2

NH2

NH2 N *

*

N

N

HO N

O

O

O

NH

O NH

Gly

HN

NH

*

*

N

N

HO

N N

O

DMA pH8.5

O

O

NH

O NH

Gly

HN

NH

L. Li et al. / Biomaterials 35 (2014) 5171e5187

OH

NH3

OH

NH3 OCH 3 O

H

OH

C

OH

OH OCH3 O

N

OH

N

OH

Folate/ DMSO/ EDC

Gly

Gly

O

NH

Gly

NH

Gly

OH

O

Gly

Gly OH O

OH O

N

OH

C

NH

H

OO

C OH

NH3

COOH O

OH

N

OH

O

NH

OH

OH OCH3 O

NH

NH

OCH3 O

OH

H

NH NH

COOH

O

OO

O

NH

OH

OH

O

O OH

OH

NH2

NH2 Fig. 1. Synthesis of doxorubicin-loaded, folate-decorated, DMA-protected HPMA copolymer (FA-APMA-DMA-Dox).

5173

5174

L. Li et al. / Biomaterials 35 (2014) 5171e5187

spectrometry using the absorbance at 492 nm (ε492 nm ¼ 80,000 M1 cm1) in 0.1 M borate buffer (pH 9) or the absorbance at 481 nm (ε481 nm ¼ 9860 M1 cm1) in deionized water, respectively. The content of APMA monomer was determined by TNBSA assay. Degradation of the DMA block coupled to the HPMA copolymers (polymers 5 and 6) was measured by the fluorescamine method [26]. Each sample of DMAcoupled HPMA copolymers was incubated with the buffer solutions of different pH values (pH 6.8 and pH 7.4) at 37  C. At specified time (0, 15, 30, 60, 120, 240 min), 100 mL of each sample was mixed with 20 mL of fluorescamine solution in DMF (2 mg/mL) and incubated in dark at room temperature for 10 min. Then the fluorescence intensity (Fs) was measured at the excitation wavelength of 365 nm and the emission wavelength of 475 nm by fluorospectrophotometer (RF-5301 PC, SHIMADZU, Japan). 100% of exposed amine (F0) was calculated from the fluorescence of the sample after the incubation in 0.1 M HCl for 24 h and the fluorescence of blank buffer solution was considered to be 0% (Fc) as a negative control. The degradation rate of DMA block was evaluated by the amount of exposed amine calculated by (Fs e Fc)/(F0  Fc)  100%. The zeta potential changes of various copolymers (0.2 mg/mL, polymers 1e6) incubated in PBS (0.01 M) at pH 6.8 or pH 7.4 at 37  C were measured by Malvern Zetasize NanoZS90 (Malvern Instruments Ltd., Malvern, UK) at predetermined time intervals (0, 5, 15, 30, 45, 60, 90, 120, 240, 480 min). All measurements were performed in triplicate. For measurement of drug release profile, 300 mL of Dox-loaded conjugate samples (equivalent Dox concentration: 10 mg/mL, polymers 13e16) were transferred into the dialysis membrane (MWCO 2000) and dialyzed against 15 mL of PBS buffer at pH 7.4, 6.8 or acetate buffer at pH 5. At predetermined time (0.5, 1, 3, 5, 7, 9, 11, 24, 36, 48 h), 100 uL of external buffer was collected and replaced by the fresh buffer. The concentration of Dox (excitation at 485 nm and emission at 590 nm) was determined by Varioskan Flash (Thermo Fisher Scientific, MA, USA). 2.4. In vivo circulation study of FA-HPMA-Dox bearing different folate contents In order to select a folate content that would not significantly affect the long circulation ability of HPMA carriers, in vivo pharmacokinetics of Dox-loaded HPMA conjugates bearing different folate contents were conducted according to previous report [35]. SD rats were randomly divided into five groups and intravenously injected with Dox, HPMA-Dox conjugates, Dox-loaded HPMA conjugates (polymers 17e19) with 5.1 mol%, 10.5 mol%, 16 mol% of folate, respectively, at Dox dose of 10 mg/kg. After administration, approximately 0.3 mL of blood was collected at 1, 3, 6, 12, 24, 48, 72 h. Plasma samples were harvested by immediate centrifugation at 3000 rpm for 10 min and store at 20  C for analysis. To precipitate plasma protein, 400 uL methanol was added to 100 uL plasma, followed by vortex for 1 min standing at 20  C for 30 min, and centrifugation at 10,000 rpm for 5 min. After that, 100 mL of the supernatant was added to a black 96-well plate and fluorescence intensity (Ex/Em: 485/590) was measured using a microplate reader.

inhibitor, caveolae endocytosis inhibitor and macropinocytosis inhibitor, respectively. To investigate the effect of temperature on cellular uptake, the experiment was also performed at 4  C. In addition, the control samples were conducted at 37  C for 2 h without any treatment, and the results of the inhibition tests were presented as the percentage of that internalized in control. To test the binding efficiency of the folate attached to neutral FA-HPMA (polymer 3) and cationic FA-APMA (polymer 4), indirect immunofluorescence by flow cytometry was performed [13]. First, cells were gently removed from plates by trypsination. Cells were then resuspended and incubated with primary antibody MAb MOv18 (dilution ¼ 1:20) and folate-bearing conjugates or free folate (at an equivalent folate content of 30 mg/mL) in a total volume of 80 mL blocking solution at 4  C in dark for 1.5 h to compete for binding the folate receptor. Then the solution was eliminated by centrifugation and the cells were washed by cold BSA (3%) containing PBS solution. 100 mL of the FITC-conjugated goat anti-rabbit IgG (H þ L) secondary antibody (dilution ¼ 1:40) in PBS was added and further incubated for 30 min. After being washed by cold PBS, cells were suspended in 300 mL PBS followed by flow cytometric analysis immediately. 2.8. Cellular uptake evaluation of Dox-loaded conjugates and their intracellular fate by confocal laser scanning microscopy Hela cells were seeded on the coverslip in 24-well plates at a density of 2  104 cells/well and incubated for 24 h at 37  C. For cellular internalization observation, cells were incubated with the Dox-loaded conjugates (polymers 13e16) at an equivalent Dox concentration of 10 mg/mL in fresh culture medium at pH 7.4 or 6.8, respectively. After incubation for 2 h at 37  C, cells were washed twice with icecold PBS and fixed with fresh 4% paraformaldehyde for 15 min at room temperature. The coverslips were then mounted on glass microscope slides with a drop of antifade mounting medium. The cellular uptake was visualized under confocal laser scanning microscopy (Zeiss Ism 510 due, BD). For subcellular localization observation, after incubation with FA-APMA-DMADox (polymer 16) at an equivalent Dox concentration of 10 mg/mL in fresh culture medium at pH 7.4 or 6.8 for 0.5, 2, 6, 12 h, cells were washed twice with ice-cold PBS and fixed with fresh 4% paraformaldehyde for 15 min at room temperature. The cells were counterstained with DAPI for cell nucleus and Lysotracker Green for acidic organelles following the manufacturer’s instructions. The coverslips were treated the same as above and subjected to CLSM observation. 2.9. Cytotoxicity assay

Hela cells and A549 cells were cultured in complete Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% FBS and penicillin (100 U/ml)/streptomycin (100 U/ml) at 37  C in a 5% CO2/95% air atmosphere. Cells were subcultured by disaggregating with Trypsin (0.25%, w/v)-EDTA (0.02%, w/v) in PBS (pH 7.4). All experiments were performed on cells in the logarithmic growth phase.

The cytotoxicity of Dox-loaded conjugates against Hela and A549 cells was evaluated by MTT assay. The cells were seeded onto 96-well plates at a density of 7  103 cells/well and incubated for 24 h. After the treatment with Dox-loaded conjugates (polymers 13e16) for 12 h or 24 h at 37  C, the mediums were removed and cells were washed twice with ice-cold PBS. Then cells were further incubated with fresh medium at pH 7.4 for another 24 h at 37  C. Afterward, the cells were incubated with MTT (5 mg/mL, 20 ml per well) for 3 h. Then the supernatant was removed before adding dimethyl sulfoxide (150 mL per well) into the wells to dissolve the formazane of MTT. The absorption at 570 nm was recorded with an ELISA plate reader (Bio-Rad, Microplate Reader 550), and the cell viability was calculated according to the following equation: (TeB)/(CeB)  100%, where T is the absorption value of the treatment group; C is the absorption value of the control (untreated) group; B refers to the absorption value of the culture medium.

2.6. Cellular uptake study

2.10. Cell apoptosis assay

2.6.1. Quantitative study of uptake by flow cytometry Hela and A549 cells were seeded on 12-well plates at a density of 1  105 cells/ well. After incubation for 24 h, the cells were treated with various FITC-labeled HPMA copolymer conjugates (0.2 mg/mL, polymer 7e12) at 37  C for 2 h. After incubation, medium was removed and cells were harvested. The samples were washed with PBS for three times followed by flow cytometric analysis immediately. The mean fluorescence intensity of 1  104 cells was recorded for each sample.

Cell apoptosis was examined by using DAPI staining method. Hela cells were cultured with Dox-loaded conjugates (polymers 13e16) at an equivalent Dox concentration of 10 mg/mL for 24 h at pH 7.4 and 6.8. Then cells were further incubated with fresh medium at pH 7.4 for another 24 h and were stained with 5 mg/mL DAPI for 5 min to visualize cell nucleus using inverted fluorescence microscopy (XD-RFL, Sunny Optical Technology). Meanwhile, a DNA fragmentation assay was also performed. DNA were firstly extracted according to the instruction of cell apoptosis DNA ladder isolation kit, followed by identification via gel electrophoresis with 1.5% agarose gel.

2.5. Cell culture

2.6.2. Internalization assay by inverted fluorescence microscopy Various FITC-labeled HPMA copolymer conjugates (0.2 mg/mL, polymer 7e12) were incubated with Hela and A549 cells at 37  C for 2 h. Then cells were washed with PBS for three times. DAPI (10 mg/mL) was added to visualize the nuclei 10 min before imaging. Cell fluorescent imaging was performed using inverted fluorescence microscopy (XD-RFL, Sunny Optical Technology). 2.7. Cellular uptake mechanisms assays in Hela cells To compare the difference among the possible internalization mechanisms employed by various HPMA conjugates by Hela cells, the cells were incubated at 4  C or pre-treated with selective inhibitors of different internalization pathways at 37  C for 1 h. Next, the indicated FITC-labeled polymer solution (0.2 mg/mL) at different temperatures or in the presence of specific inhibitor was added and further incubated for 2 h. Sodium azide (1 mg/mL), excess free folic acid (1 mM), chlorpromazine (10 mg/mL), filipin (1 mg/mL), amiloride (0.3 mg/mL) were used as active transport inhibitor, folate-receptor mediated endocytosis competitor, clathrin endocytosis

2.11. Inhibition of tumor spheroids growth For multicellular tumor spheroids formation, Hela cells were seeded in a 96-well plate pre-coated with 2% (w/v) agarose gel at the density of 1000 cells/well. After being cultured for 4 days, the uniform and compact spheroids were selected and treated with Dox-loaded conjugates (polymers 13e16) at an equivalent Dox concentration of 10 mg/mL for 7 days. FA-APMA-DMA-Dox (polymer 16) and APMADMA-Dox (polymer 15) were pre-incubated at pH 6.8 for 4 h. The size of tumor spheroids was carefully observed under the inverted fluorescence microscopy (XDRFL, Sunny Optical Technology). Growth inhibition was calculated with the following formula: V ¼ (p  dmax  dmin)/6, where dmax is the maximum diameter and dmin is the minimum diameter of each spheroid. The change ratio of tumor spheroid volume was calculated with the following formula: ratio% ¼ (Vday i/ Vday0)  100%, where Vday i is the tumor spheroid volume on the ith day after applying drug, and Vday0 is the tumor spheroid volume prior to treatment.

L. Li et al. / Biomaterials 35 (2014) 5171e5187

3.1. Polymer synthesis and characterization A series of HPMA copolymers containing different types and amount of functional groups were synthesized by varying the feeding ratio of positively charged APMA comonomer and folate targeting ligand, which contained the neutral copolymers (HPMA, FA-HPMA), positively charged copolymers (APMA, FA-APMA) and charge-shielded copolymers (APMA-DMA, FA-APMA-DMA) with or without folate decoration. Their corresponding FITC-labeled and doxorubicin-loaded derivatives were also synthesized for different purposes. The characteristics including molecular weight (Mw), poly dispersity index (PDI), folate content, FITC content and drug loading were summarized in Table 1. And as illustrated in Fig. 1, the doxorubicin-loaded, folate-bearing, DMA-protected HPMA copolymer (FA-APMA-DMA-Dox, polymer 16) were generated by radical copolymerization of HPMA and positively charged APMA comonomers with a polymerizable drug derivative MA-GGNHN ¼ Dox, followed by the folate modification and DMA protection. The molecular weight of the conjugate was 39.5 KDa with poly dispersity index of 1.73. The folate content and Dox load were 4.66 and 10.9 wt%, respectively. Here, doxorubicin as the model drug was conjugated through the pH-sensitive hydrazone linkage which could ensure the prompt drug release in the endo/lysosome after endocytosed into the cells. The APMA segment containing the primary amino groups in the drug carrier backbone not only served as cationic pendants, but also provided conjugation sites for folic acid. Thereby partial amino groups were modified by certain amount of folates and the remaining amino groups were further reacted with the excess DMA to mask the positive charge at neutral pH. The resultant b-carboxylic acid amides have been reported to be acid-labile and could degrade quickly under slightly acidic environmental conditions in tumor as the result of nucleophilic catalysis by the carboxylic acid [26e28]. 3.2. In vivo circulation studies of FA-HPMA-Dox bearing different folate content Despite that increasing overall folate density on the nanoparticle surface or in the polymeric system could result in increased cell Table 1 Characteristics of the synthesized HPMA copolymers containing different functional groups. Polymer no.

HPMA copolymers

Mw (kDa)

PDI

Mol% folate

Wt% FITC

Wt% Dox

Mol% APMA monomer

1 2 3 4 5 6 7 8 9 10 11 12

HPMA APMA FA-HPMA FA-APMA APMA-DMA FA-APMA-DMA HPMA-FITC APMA-FITC FA-HPMA-FITC FA-APMA-FITC APMA-DMA-FITC FA-APMA-DMAFITC HPMA-Dox FA-HPMA-Dox APMA-DMA-Dox FA-APMA-DMADox 5.1%FA-P-Dox 10.5%FA-P-Dox 16%FA-P-Dox

32.9 35.8 38.3 40.5 36.4 41.6 25.1 25.8 26.7 28.7 28.4 30.6

1.48 1.46 1.43 1.65 1.45 1.51 1.57 1.42 1.57 1.59 1.66 1.64

e e 4.71 4.03 e 4.10 e e 3.37 3.70 e 3.74

e e e e e e 2.73 2.64 2.31 2.51 2.33 2.32

e e e e e e e e e e e e

e 26.32 e 22.15 e e e 25.41 e 21.58 e e

27.5 37.1 34.1 39.5

1.83 1.68 1.57 1.73

e 5.34 e 4.66

e e e e

17.2 11.5 17.7 10.9

e e e e

34.2 34.7 36.2

1.55 1.68 1.72

5.10 10.5 16.0

e e e

11.6 11.3 11.2

e e e

13 14 15 16 17 18 19

internalization in vitro, in vivo circulation times were demonstrated to be compromised by the inclusion of this hydrophobic moiety, thereby offsetting the benefits of active targeting [17,18]. So the pharmacokinetics of similar molecular weighed Dox-loaded HPMA conjugates (polymers 17e19) with 5.1 mol%, 10.5 mol%, 16 mol% of folate were evaluated to determine the effect of different folate contents on the long circulation ability of HPMA carriers. In accordance with previous studies, our results showed HPMA-Dox exhibited prolonged blood residence time compared with free Dox (Fig. 2). As the folate content increased, the blood clearance of the drug also increased. The decreased water solubility of the copolymers and increased recognition by macrophages might probably account for this limitation [17,18]. At 72 h, plasma concentrations of both 10.5% FA-HPMA-Dox (polymer 18) and 16% FA-HPMA-Dox (polymer 19) were similar to that of the free Dox, whereas 5.1% FAHPMA-Dox (polymer 17) showed comparable drug elimination level with non-modified HPMA-Dox (polymer 13). As the degree of EPR effect is greatly dependent on the nanocarrier circulation time and the passive accumulation in tumor would be a prerequisite for active targeting, Dox-loaded HPMA conjugate bearing about 5 mol% of folate was therefore selected for the subsequent in vitro study, even though the HPMA copolymers with higher content of folate might potentially increase the cell uptake efficiency. 3.3. Effect of positive charge and folate moiety on the cellular uptake of HPMA copolymers To identify the role of positive charge and folate each played in the internalization of modified HPMA copolymers, the two modification strategies used alone or combined were compared at both pH 6.8 and 7.4 utilizing two different cell lines, the folate-receptor positive Hela cell and folate-receptor negative A549 cell as determined by immunofluorescence assay in Fig. 3A and C. The cell internalization of these copolymers seemed to be pH-independent, for the uptake at both pHs showed similar trends. When the neutral HPMA-FITC (polymer 7) was taken as a reference, results from Fig. 3B and E showed that the cell uptake of folate modified FA-HPMA-FITC (polymer 9) had only marginal uptake improvement of approximately 2-fold in Hela cells (p < 0.05), while the positively charged APMA-FITC (polymer 8) increased 8.8-fold (p < 0.05), which suggested that the electrostatic interaction with negatively charged cell membrane might have greater impact on the

100

μg Dox/mL plasma

3. Results and discussion

5175

Dox HPMA-Dox 5.1% FA-HPMA-Dox 10.5% FA-HPMA-Dox 16% FA-HPMA-Dox

10

1 0

10

20

30 40 50 Time (h)

60

70

80

Fig. 2. Plasma concentration of doxorubicin after intravenous administration of free doxorubicin (Dox), non-targeted HPMA-Dox, folate targeted 5.1 mol% FA-HPMA-Dox, 10.5 mol% FA-HPMA-Dox and 16 mol% FA-HPMA-Dox at the dose of 10 mg Dox/kg.

5176

Count 0 0

103 104 105 106 Comp-FL1::FL1 Log

32 30 28 26 24 22 20 18 16 14 12 10 8 6 4 2 0

**

**

APMA-FITC FA-APMA-FITC HPMA-FITC FA-HPMA-FITC

**

C

APMA-FITC

Sample Name Control.LMD

A549.LMD

*

*

0 pH6.8

0 103 104 105 106 Comp-FL1:FL1 Log

pH7.4 Hela cells

E

D

FA-APMA-FITC HPMA-FITC

FA-HPMA-FITC

F

pH 6.8

pH 6.8

pH 7.4

pH 7.4

APMA-FITC

36 34 32 30 28 26 24 22 20 18 16 14 12 10 8 6 4 2 0

**

**

pH6.8

APMA-FITC FA-APMA-FITC HPMA-FITC FA-HPMA-FITC

pH7.4 A549 cells

FA-APMA-FITC HPMA-FITC

FA-HPMA-FITC

Fig. 3. Determination of folate receptor expression by indirect immunofluorescence on Hela cells (A) and A549 cells (C) using flow cytometry after cells were trypsinized and labeled with anti-folate receptor primary antibody (Mov18, 1:40) followed by FITC-conjugated secondary antibody. Nonspecific fluorescence was assessed using the secondary antibody only (control group). The results are representative of two independent experiments; Quantitative study of uptake of copolymers by flow cytometry in Hela cells (B) and A549 cells (D) after 2 h incubation at pH 6.8 and 7.4. The results were expressed as bar chart (n ¼ 3, *p < 0.05, **p < 0.01); Internalization of copolymers by Hela cells (E) and A549 (F) after 2 h incubation at pH 6.8 and 7.4 imaged by inverted fluorescence microscopy.

L. Li et al. / Biomaterials 35 (2014) 5171e5187

Flourescence intensity

Sample Name Hela.LMD Control.LMD

**

Flourescence intensity

B

Count

A

control

control

1.2 Internalization(% of control )

0.8 4°C NaN3

NaN3

4°C

4°C

NaN3

*

*

*

0.4

*

*

*

0.2 0.0

1.0

*

0.8

*

0.6

*

0.4 0.2 0.0

0.6 0.4 0.2 0.0

0.6

*

*

0.4 0.2 0.0

pH6.8

pH7.4 APMA-FITC

pH6.8

pH7.4 FA-HPMA-FITC

* *

pH6.8

0.8

pH7.4

*

*

Control

Amiloride

*

*

Folate

*

Filipin

*

Folate

APMA-FITC

Chlorpromazine

Folate

Chlorpromazine

*

1.0 Amiloride

*

*

pH7.4

FA-APMA-FITC

E

Filipin

*

Control

Amiloride

Chlorpromazine

Control 0.8

*

Folate

*

*

1.0

Filipin

*

Folate

Filipin

Amiloride

Chlorpromazine

Control

Folate

Amiloride

Filipin

*

Internalization(% of control )

0.8

Chlorpromazine

1.0

Control

Internalization(% of control )

1.2

D

pH6.8

Amiloride

pH7.4

FA-HPMA-FITC

Filipin

pH6.8

Chlorpromazine

APMA-FITC

Control

FA-HPMA-FITC FA-APMA-FITC

C

*

*

L. Li et al. / Biomaterials 35 (2014) 5171e5187

Internalization(% of control )

1.0

0.6

control free folate

B

control

Internalization(% of control )

A

*

0.6 0.4 0.2 0.0 pH6.8

pH7.4 FA-APMA-FITC

Fig. 4. Cell endocytosis mechanism assay. (A) Energy-dependent cell uptake at 4  C and in the presence of NaN3. (B) Cell-specific endocytosis inhibited by free folate competition. Uptake of (C) APMA-FITC, (D) FA-HPMA-FITC, (E) FAAPMA-FITC by Hela cells when pretreated with chlorpromazine, filipin and amiloride for 1 h. Fluorescence intensity in the non-inhibited cells was used as control (*p < 0.05 vs. control).

5177

5178

L. Li et al. / Biomaterials 35 (2014) 5171e5187

enhancement of cell uptake than the hydrophobic folate ligande receptor mediated interaction in this case. The limited uptake increased by FA-HPMA-FITC (polymer 9) might be probably due to the inaccessibility of hydrophobic folate to receptors on cell surface, which was reported by Ref. [14]. In addition, when the two strategies were combined, the strongest fluorescence intensity with about 13.5-fold increase over HPMA-FITC was found in FA-APMA-FITC (polymer 10), indicating that the dual-targeting approach could exhibit a synergic effect on cell uptake. Interestingly, this synergic effect was even superior to the overall rise of single-modified FAHPMA-FITC (polymer 9) and APMA-FITC (polymer 8), suggesting that the enhancement of cell uptake by FA-APMA-FITC (polymer 10) was just more than the overall effect of cationic charge and folate working separately. Hypothetically, the positive charge could probably amplify the targeting efficiency of folate in HPMA polymer chain, which was investigated and discussed in later study. As the incorporation of hydrophobic groups into HPMA copolymers might give rise to enhanced uptake [36], the insoluble folate may just increase the cell uptake through hydrophobic interaction with a lipophilic part of cell membrane but not via receptor recognition. Therefore, the cell uptake by a negative control of folate receptor deficient cell line A549 was also evaluated. In contrast to results obtained by the Hela cells, the folate modified copolymers showed comparable or even lower internalization into the A549 cells compared with that of the non-folate-modified ones (Fig. 3D and F). The results demonstrated that the cell uptake enhancement by Hela cells after folate modification was due to the ligandereceptor mediation but not hydrophobic interaction. The lower but not significant internalization of FA-APMA-FITC (polymer 10) in A549 cells may be owing to lower zeta potential level of the polymers after FA modification. On the other hand, the folatemediated endocytosis of FA-HPMA-FITC (polymer 9) and FAAPMA-FITC (polymer 10) by Hela cells were thus verified. 3.4. Cellular uptake mechanisms in Hela cells In order to understand how each modification (folate decoration or electrostatic force) influence the cell uptake, the cell entry pathways of single or dual-modified copolymers were investigated. Thereby, systematic cellular uptake mechanisms of APMA-FITC (polymer 8), FA-HPMA-FITC (polymer 9) and FA-APMA-FITC (polymer 10) were performed. As shown in Fig. 4A, either the presence of sodium azide, a metabolic inhibitor to deplete ATP, or low temperature of 4  C which maintains the energy metabolism of cells at low level, dramatically decreased the cell uptake of all tested copolymers (p < 0.05), suggesting that their uptake mechanisms occurred through energy-dependent endocytosis. Then the folate receptor-mediated endocytosis was directly examined by the competition of excess free folate. The lack of attached folate in APMA-FITC (polymer 8) led to no significant decrease in cell uptake expectedly. And apparent inhibition to around 50% of control in cell uptake was achieved in FA-HPMA-FITC (polymer 9), confirming the folate receptor-mediated endocytosis was involved. Interestingly, only 20% cellular uptake reduction (p < 0.05) was observed by FA-APMA-FITC (polymer 10) with the addition of free folate ligands even though they had the highest internalization efficiency (Fig. 4B). This phenomenon implied that in addition of receptor-mediated endocytosis, other endocytic pathways may also take part in the cell uptake of FA-APMA-FITC (polymer 10). To further investigate the endocytic pathways of these copolymers with more details, selective inhibitors of chlorpromazine, filipin and amiloride were utilized [19]. Amiloride, an inhibitor of macropinocytosis, blocked the cell uptake for all tested conjugates to different extents (Fig. 4C, D and E, p < 0.05), suggesting that macropinocytosis played an important role in the uptake process of

macromolecular carriers. For APMA-FITC (polymer 8), the cell uptake was largely suppressed by chlorpromazine (p < 0.05), a selective inhibitor of clathrin-mediated endocytosis, but not by filipin, an inhibitor of caveolae-mediated endocytosis (Fig. 4C). This finding was agreed by the study of [19] that positively charged HPMA copolymers were mainly uptaken by clathrin-mediated endocytosis. For FA-HPMA-FITC (polymer 9), both chlorpromazine and filipin could decrease its uptake (p < 0.05), but with more significant reductive extent achieved by the filipin (Fig. 4D), in line with some folate modified drug delivery systems that were mainly uptaken by caveolae-mediated endocytosis [37e39]. The caveolae-mediated endocytosis was probably related to receptor-mediated endocytosis derived from folic acid molecule that is internalized by caveolae-flask-like invaginations on the cell surface that bud from membrane microdomains rich in cholesterol and protein cavelion [40]. When it came to cell entry of FA-APMA-FITC (polymer 10), various endocytic pathways including clathrin-mediated endocytosis, caveolae-mediated endocytosis and macropinocytosis were vigorously engaged. However, greatest decrease was observed when treated with chlorpromazine (p < 0.05), indicating electrostatic absorptive endocytosis governed by clathrin-mediated endocytosis contributed more in the cell uptake of dual-modified FA-APMA-FITC (polymer 10) than the folate receptor-mediated endocytosis governed by caveolae-mediated endocytosis, which was consistent with the results obtained in Fig. 3. We could also come to a conclusion that the synergic enhanced effect on cell uptake of dualmodified FA-APMA-FITC (polymer 10) could be ascribed to the increased endocytic pathways employed when internalizing. Another interesting phenomenon observed in Fig. 4 was that when HPMA-FITC (polymer 7) was taken as control, the increased amount of internalized copolymers achieved by FA-APMA-FITC (13.5-fold for polymer 10) was even larger than total increased amount achieved by APMA-FITC (8.8-fold for polymer 8) plus FAHPMA-FITC (2-fold for polymer 9). And compared with other folate targeting systems, the folate used alone in this study exhibited minor effect on cell uptake. So it was speculated that the positive charge could actually facilitate the folate binding and maximize its targeting potential. As folate is a hydrophobic group, it may be buried inside the neutral hydrophilic polymer chain, which may hamper the binding and lead to targeting efficiency loss by interfering the ligandereceptor contact (Fig. 5A). This is supported by several reports that used different drug carriers [14e16]. On the other hand, the positive charge of the copolymer backbone would first adhere to the cell surface by electrostatic attraction (Fig. 5B). This could in turn bring the attached folate in close contact with the membrane. Then the chance of folate interacting with the receptor would be greatly increased, leading to an enhanced targeting ability exerted by the folate (Fig. 5C). To prove this hypothesis, the binding efficiency of FA-HPMA (polymer 3) and FA-APMA (polymer 4) to the folate receptor on Hela cell was tested in competition with antiFR primary monoclonal antibody Mov-18 followed by a FITCconjugated secondary antibody. As shown in Fig. 6, the antibody alone bound to 84.01% of tested cells leading to the strongest fluorescence, while the percentage of antibody-bounded cell decreased significantly to 6.17%, 34.0%, 71.2% when co-cultured with free folate, FA-APMA (polymer 4), FA-HPMA (polymer 3). Moreover, the amount of folate antibody that specifically bound to the folate receptor was inhibited to greater extent in competition with FA-APMA (polymer 4) than FA-HPMA (polymer 3), thus confirming that the folate binding affinity of cationic FA-APMA (polymer 4) was augmented compared with that of neutral FA-HPMA (polymer 3). In summary, in terms of dual-modified FA-APMA (polymer 4) internalization, the enhanced cellular uptake could be ascribed to the increased endocytic mechanisms, and electrostatic attraction played a dominant role. The positive charge not only enhanced cell

L. Li et al. / Biomaterials 35 (2014) 5171e5187

5179

Fig. 5. Schematic illustration of cell internalization mechanism of (A) FA-HPMA, (B) APMA and (C) FA-APMA. The hydrophobic folate may be buried inside the neutral hydrophilic FA-HPMA polymer chain and not be accessible to the folate receptor on the cell surface while the cationic APMA could efficiently enter into cells by interacting with the negatively charged membrane. Therefore, the positively charged FA-APMA could first adhere to the cell surface by electrostatic attraction, which in turn could bring the attached folate in close contact with the membrane. Then the chance of folate binding to the receptor would be greatly increased due to the close proximity of copolymer to the surface of target cell, leading to an enhanced targeting ability exerted by the folate.

uptake by triggering absorptive endocytosis, but also facilitated folate binding to its receptor, thus resulting in a synergistic effect on the cell internalization. 3.5. Tumor extracellular acidity-triggered charge reversion In order to avoid non-specific binding in the circulation, the shielding group 2,3-dimethylmaleic anhydride (DMA) was therefore introduced to mask the positive charge at pH 7.4 but regenerate it at pH 6.8 [26e28]. To verify the pH-dependent hydrolysis of the DMA modified amide bonds, the degradation of the DMA groups were monitored by measuring the amount of the re-exposed primary amine using fluorescamine method after incubation of the copolymers (polymers 5 and 6) at pH 7.4 and 6.8, mimicking the physiological environment and tumor microenvironment, respectively. As shown in Fig. 7A, the cleavage of coupled DMA in FAAPMA-DMA (polymer 6) rapidly reached approximately 50% within 30 min at pH 6.8 and further increased to more than 80% with a prolonged incubation time of 2 h, whereas the hydrolysis profile was relatively minor and slow at pH 7.4 reaching a plateau of only 30% DMA release in 4 h. Similar trends were also observed in the case of APMA-DMA (polymer 5), indicating a desirable pH-responsive detachment of DMA as the shielding group. Thus the much faster hydrolysis rate of the coupled anionic DMA at pH 6.8 was expected to endow the charge-shielded copolymers with charge-switchable ability in slightly acidic environment. As confirmed in the Fig. 7B, APMA-DMA (polymer 5) and FA-APMADMA (polymer 6) were both initially negatively charged at pH 6.8, but underwent a sharp charge reverse to become positively charged within 30 min and continuously rose to a plateau with positive zeta potentials of above 10 mV within 2 h, just slightly lower than the unshielded APMA (polymer 2) and FA-APMA (polymer 4), probably due to the incomplete DMA detachment, which was in consistence with the results obtained in Fig. 7A. By comparison, the zeta potentials of the charge-shielded copolymers (polymer 5 and 6), with an increase at such a slight and slow rate, still remained negative even after 4 h incubation at pH 7.4 as a consequence of the presence of the unreleased DMA (Fig. 7C). All these results validated that with the aid of tumor extracellular pH-activated degradation of DMA, an immediate charge reverse intended for efficient entry by tumor cells through absorptive interaction could be attained by the DMAshielded HPMA copolymers. 3.6. pH-responsive drug release After the drugs are escorted into the cancerous cells by the conjugates, the intracellular release of the original drugs from the

carrier is considered a prerequisite for restoring the anticancer activity and subsequent cytotoxicity [34,41] So the Dox release profiles of different HPMA copolymers were investigated under different pH conditions. As expected, the hydrazone spacers of all conjugates (polymers 13e16) exhibited relative stability at pH 7.4 and 6.8 with no more than 20% of Dox release after 24 h (Fig. 8), which could be advantageous in reducing the premature release of Dox extracellularly. As the pH declined to 5, a rapid drug release could also be observed for all conjugates. Nevertheless, it should be noted that a slightly decrease in Dox release rate of folate modified copolymers FA-HPMA-Dox and FA-APMA-DMA-Dox (both with about 70% Dox release after 48 h at pH 5) took place (Fig. 8B and D) in comparison with HPMA-Dox (94% of Dox released after 48 h) at pH 5. Probably the steric hindrance and hydrophobic interactions in the hydrophilic polymer chain after introducing the waterinsoluble folate groups were responsible for the loss of Dox release rate, which was demonstrated by the Karel Ulbrich’s group that the presence of the hydrophobic substituent in the HPMA copolymers could lead to a decrease in the Dox release rate of w20% [42]. However, this Dox release rate was still fast enough to guarantee high therapeutic intracellular Dox concentration. Therefore, in addition with the tumor extracellular pH sensitivity to enhance the cell uptake, a second pH-responsiveness at endo/lysosomal level to ensure drug release was also achieved. 3.7. pH-dependent cell uptake and intracellular trafficking To further demonstrate whether the charge reverse ability and folate functionality could biologically bring about the enhancing effect on cellular internalization, the cell uptake of various drug carriers were tested on folate receptor over-expressed Hela cells by flow cytometry and fluorescence microscopy first. As shown in Fig. 9A and B, remarkably more efficient uptake occurred in chargeswitchable APMA-DMA-FITC (polymer 11) and FA-APMA-DMAFITC (polymer 12) at pH 6.8, whereas much weaker fluorescence intensity was observed at pH 7.4. Given that cell membranes are generally negatively charged, these findings indicated that the electrostatic repulsion at pH 7.4 in the circulation between the DMA-coupled copolymers and cell membranes could minimize the cell uptake in the normal tissues, while the electrostatic attraction of the copolymers to cell membrane took place at pHe due to the stimulated hydrolysis of positive charge-shielded groups, thus enhancing the cellular internalization. And in particular, stronger fluorescence was detected for FA-APMA-DMA-FITC (polymer 12) than APMA-DMA-FITC (polymer 11) in Hela cells at pH 6.8 (p < 0.05), while both copolymers showed comparable internalization into the A549 cells, thus indicating the specific role of folate.

5180 L. Li et al. / Biomaterials 35 (2014) 5171e5187 Fig. 6. Folate binding efficiency assay by indirect immunofluorescence. Nonspecific fluorescence was assessed using the secondary antibody only as control group (A). Anti-FR primary monoclonal antibody Mov-18 were co-incubated with (B) free folate, (C) FA-APMA, (D) FA-HPMA, (E) blank in Hela cell suspension to compete for the folate receptor, followed by labeling with FITC-conjugated secondary antibody. The samples were analyzed by flow cytometry by measuring the relative fluorescence intensity of FITC per cell at the FITC channel (F).

A

B

C

#

#

80 60

#

50

*

*

#

70

* *

40

*

30

APMA-DMA pH7.4 APMA-DMA pH6.8 FA-APMA-DMA pH7.4 FA-APMA-DMA pH6.8

20 10 0

30

60

90 120 150 180 210 240 Time (min)

16

HPMA APMA APMA-DMA

FA-HPMA FA-APMA FA-APMA-DMA

12 8 4 0 -4 -8 -12

HPMA APMA APMA-DMA

-16 -20

0

0

30 60 90 120 150 180 210 240 450 480 Time (min)

FA-HPMA FA-APMA FA-APMA-DMA

30 60 90 120 150 180 210 240 450 480 Time (min)

Fig. 7. (A) The degradation of DMA modified amide at pH 7.4 and pH 6.8 (*p < 0.05 vs. APMA-DMA pH 7.4, #p < 0.05 vs. FA-APMA-DMA pH 6.8). (B) Zeta potential variation of the copolymers exposed at pH 6.8 for different time periods. (C) Zeta potential variation of the copolymers exposed at pH 7.4 for different time periods.

0.6 0.4 0.2 0.0

0

10

20 30 Time (h)

40

50

pH5 pH6.8 pH7.4

0.8 0.6 0.4 0.2 0.0 0

10

20 30 Time (h)

40

50

D 1.0

pH5 pH6.8 pH7.4

0.8

Released Dox of total Dox (%)

0.8

C

1.0

Released Dox of total Dox (%)

B pH5 pH6.8 pH7.4

Released Dox of total Dox (%)

Released Dox of total Dox (%)

A 1.0

0.6 0.4 0.2 0.0 0

10

20 30 Time (h)

40

50

1.0

L. Li et al. / Biomaterials 35 (2014) 5171e5187

0

20

20 16 12 8 4 0 -4 -8 -12 -16 -20

Zeta potential (mv)

90

Zeta potential (mv)

(Fs-Fd.w)/(F0-Fd.w) X100%

100

pH5 pH6.8 pH7.4

0.8 0.6 0.4 0.2 0.0

0

10

20 30 Time (h)

40

50

Fig. 8. Release profiles of Dox from (A) HPMA-Dox, (B) FA-HPMA-Dox, (C) APMA-DMA-Dox, (D) FA-APMA-DMA-Dox incubated at pH 5.0, pH 6.8 and pH 7.4 buffer.

5181

5182

L. Li et al. / Biomaterials 35 (2014) 5171e5187

B **

24 22 20 18 16 14 12 10 8 6 4 2 0

APMA-DMA-FITC FA-APMA-DMA-FITC

APMA-DMA-FITC

FA-APMA-DMA-FITC Flourescence intensity

Flourescence intensity

A

pH 6.8

*

pH6.8

pH7.4

24 22 20 18 16 14 12 10 8 6 4 2 0

pH7.4

C

APMA-DMA-Dox

FA-APMA-DMA-Dox

FA-APMA-DMA-FITC

pH 6.8

pH7.4

pH6.8

Hela cells

APMA-DMA-FITC

APMA-DMA-FITC FA-APMA-DMA-FITC

pH7.4 A549 cells

HPMA-Dox

FA-HPMA-Dox

pH 6.8

pH 7.4

D

30min

2h

6h

12h

E

DAPI

DAPI

Lysosome

Lysosome

Dox

Dox

Merge

Merge

30min

2h

6h

12h

Fig. 9. pH-dependent cell uptake by Hela cells (A) and A549 cells (B). Internalization of Dox-loaded copolymers by Hela cells at pH 6.8 and pH 7.4 imaged by confocal laser scanning microscopy (C). Intracellular fate of FA-APMA-DMA-Dox incubated at pH 6.8 (D) and pH 7.4 (E) for different time periods. (n ¼ 3, *p < 0.05, **p < 0.01).

Despite no enhancing effect of folate on the uptake by A549, FAAPMA-DMA-FITC (polymer 12) or APMA-DMA-FITC (polymer 11) still displayed excellent tumor extracellular pH selectivity, which was barely visible in the cells at pH 7.4 but showed tremendous increase of fluorescence at pH 6.8. Similar results were also obtained in the cellular uptake of Doxloaded conjugates (polymer 13e16) in Hela cell (Fig. 9C), with FAAPMA-DMA-Dox (polymer 16) showing the brightest fluorescence incubated at pH 6.8. Then different intracellular fates of FA-APMA-

DMA-Dox (polymer 16) at pH 6.8 and pH 7.4 were compared (Fig. 9D and E). At pH 7.4, no detectable fluorescence for Dox was observed at 30 min of incubation. As incubation time prolonged, relatively weak red fluorescence for Dox was seen overlaid with the green fluorescence for endo/lysosomes at 2 h and 6 h. And only a small portion of Dox entered the nucleus where they took anticancer actions at 12 h (Fig. 9E). In contrast, at pH 6.8 the conjugates that already gained positive charge in response to the first stage of pHsensitivity for tumor extracellular environment as early as 30 min

A

B

D

E

F

G

H

I

J

L. Li et al. / Biomaterials 35 (2014) 5171e5187

C

Fig. 10. Cell cytotoxicity of various polymeric carriers at (A) pH 7.4 and (B) pH 6.8 by MTT assay. The cell cytotoxicity of various Dox-loaded conjugates to Hela cells (CeF). Cells were treated with the conjugates at pH 7.4 (C and E) or pH 6.8 (D and F) for 12 h (C and D) or 24 h (E and F), then further incubated with fresh culture media for another 24 h, followed by MTT assay. The cell cytotoxicity of various Dox-loaded conjugates on A549 cells (GeJ). Cells were treated with the conjugates at pH 7.4 (G and I) or pH 6.8 (H and J) for 12 h (G and H) or 24 h (I and J), then further incubated with fresh culture media for another 24 h, followed by MTT assay.

5183

5184

L. Li et al. / Biomaterials 35 (2014) 5171e5187

were seen to quickly associate with the cell membrane. With the synergistic effect of absorptive and receptor-mediated endocytosis, they were trafficked to endo/lysosome much more rapidly at 2 h. Colocalization with endo/lysosome is essential for this drug delivery system, for it is the place where drug is released. But if retaining there for too long, drugs may either be exocytosed outside the cell or get deactivated by the harsh environment where a relatively high level of bioactive enzymes exist [21]. The endo/lysosomal escape caused by the proton sponge effect due to the positive charge of the conjugate is not likely to happen in this case, for the buffering ability of the primary amino group at this pH is relatively low [43]. Thus rapid drug release and subsequent endo/lysosomal escape by diffusion would be of great paramount to achieve efficient drug delivery [44]. As the second stage of pH-sensitivity started with the degradation of hydrazone in endo/lysosomes, some of the Dox were distributed outside the endo/lysosome and even entered into nucleus at 6 h. And once localized in the cytoplasm, Dox has the very tendency to accumulate in the nucleus [45]. Unsurprisingly, a great amount of Dox was finally seen to arrive at nucleus at 12 h (Fig. 9D). Therefore, a promising cytotoxicity effect of FA-APMA-DMA-Dox (polymer 16) at pHe could be anticipated. 3.8. Cytotoxicity assay and apoptosis assay To investigate whether the enhanced cell uptake by the charged-switchable, folate-modified conjugates could transform into increased anticancer activity, cell cytotoxicity assay was performed. First, cytotoxicity of various blank polymeric carriers (polymers 1e6) was evaluated. As shown in Fig. 10A and B, no cytotoxicity occurred for all conjugates except that only the positive charged copolymers at highest concentration (0.8 mg/mL) showed a minor decrease in cell viability. It should be noted that the concentration of Dox-loaded conjugates used in the later study was not higher than 0.1 mg/mL. So the carrier itself should contribute no cytotoxicity to the cells.

A

Control

FA-HPMA-Dox

Then the folate receptor over-expressed Hela cells were treated with the Dox-loaded conjugates (polymer 13e16) or free drugs at pH 6.8 and pH 7.4. After 12 h or 24 h of incubation, the conjugates or drugs were removed and cells were further incubated for another 24 h to allow for sufficient intracellular drug release. As depicted in Fig. 10CeF, all conjugates showed time-dependent and concentration-dependent toxicity towards cells, and APMA-DMADox (polymer 15) as well as FA-APMA-DMA-Dox (polymer 16) even displayed pH-dependent manner. It was found that both DMA-coupled conjugates exhibited a superior cytotoxicity at pH 6.8 over pH 7.4 regardless of the incubation time, probably due to the elevated intracellular drug concentration by the charge-reversible properties at mild acidic environment. Surprisingly, FA-HPMADox (polymer 14) showed a slightly lower cytotoxicity towards Hela cells than HPMA-Dox (polymer 13), even though the folate modified conjugate had higher cell uptake. This could be explained by the slower drug release of FA-HPMA-Dox (Fig. 8) that counteracted the effect of improved internalization which was not sufficient enough to make up for the loss of the impaired drug release profile by FA-HPMA-Dox (polymer 14). However, in the cases of charge-reversible copolymers at pH 6.8 where the positive charge maximized the targeting ability of folate and the conjugates arrived at endo/lysosome more abundantly and rapidly, FA-APMA-DMADox (polymer 16) were proved to be able to overcome the drug release setback or “folate dilemma”, and eventually were more potent in killing folate receptor positive Hela cells than APMADMA-Dox (polymer 15). Then the Hela cell was used to study the cell apotosis induced by incubating with various Dox-loaded conjugates for 24 h. The results demonstrated that both FA-APMADMA-Dox (polymer 16) and APMA-DMA-Dox (polymer 15) at pH 6.8 led to cell apoptosis, which was reflected by characteristic changes in nuclear shape including the separation of cell nuclei into segments with unshaped borders after DAPI staining while no evidence of the formation of cell nuclei fragments was shown when treated with other groups (Fig. 11A). Also severe DNA condensation

HPMA-Dox

FA-APMA-DMA-Dox APMA-DMA-Dox

pH 6.8

pH 7.4

B

a

b

c

d

e

a’

b’

c’

d’

e‘

Fig. 11. Induction of apoptosis in Hela cells after 24 h incubation with various Dox-loaded conjugates. (A) Morphological and nuclear changes of Hela cells after DAPI staining. (B) DNA fragmentation assay, a and a0 : control at pH 6.8 and pH 7.4; b and b0 : FA-HPMA-Dox at pH 6.8 and pH 7.4; c and c0 : HPMA-Dox at pH 6.8 and pH 7.4; d and d0 : FA-APMA-DMADox at pH 6.8 and pH 7.4; e and e0 : APMA-DMA-Dox at pH 6.8 and pH 7.4. Hela cells incubated with drug-free DMEM served as the control.

L. Li et al. / Biomaterials 35 (2014) 5171e5187

was obviously observed for cells treated with FA-APMA-DMA-Dox (polymer 16) at pH 6.8 (Fig. 11B). To prove that folate could indeed lead to improved cytotoxicity of charge-reversible conjugates at pH 6.8 towards folate receptor over-expressed cells, the cytotoxicity test on the folate receptor negative A549 cells was set as control. As shown in Fig. 10GeJ, the absence of targeting ligand function of folate, together with the lower zeta potential level of FA-APMA-DMA-Dox (polymer 16) resulted in its inferior anticancer activity compared with APMADMA-Dox (polymer 15). However, both conjugates again showed selectively higher apoptotic effect on cells at pH 6.8 than pH 7.4 and

0 day

1 day

2 day

3 day

5185

neutral conjugates (polymerc13 and polymer 14), indicating their feasibilities regardless of the cell types. 3.9. Inhibition of tumor spheroids growth Tumor spheroids with three-dimensional architecture were usually employed to predict the drug effect as an ideal in vitro platform mimicking solid tumors [46,47]. In this study, the inhibition of tumor spheroids growth was evaluated by measuring the size of spheroids treated with various Dox-loaded conjugates. As shown in Fig. 12, the control group (284% of original spheroid

4 day

5 day

6 day

7 day

Control

FA-APMA-DMA-Dox/pH6.8

FA-HPMA-Dox

APMA-DMA-Dox

HPMA-Dox

FA-APMA-DMA-Dox/ pH7.4

Control FA-APMA-DMA-Dox at pH6.8 FA-HPMA-Dox APMA-DMA-Dox at pH6.8 HPMA-Dox FA-APMA-DMA-Dox at pH7.4

Tumor spheroid volumn ratio (%)

3.5

3.0

2.5

2.0

*

*

* 1.5

1.0

#

#

#&

#&

#&

#&

5

6

7

0.5 0

1

2

3 4 Time (days)

Fig. 12. Inhibition of tumor spheroids growth (n ¼ 5, *p < 0.05 HPMA-Dox vs. FA-HPMA-Dox, #p < 0.05 FA-APMA-DMA-Dox at pH 7.4 vs. FA-APMA-DMA-Dox pretreated at pH 6.8, & p < 0.05 APMA-DMA-Dox pretreated at pH 6.8 vs. FA-APMA-DMA-Dox pretreated at pH 6.8).

5186

L. Li et al. / Biomaterials 35 (2014) 5171e5187

volume on 7th day) kept growing and became more compact eventually. The inhibition of spheroids growth by other groups displayed a similar trend to the results obtained in MTT assay, with the inhibition ability followed the order: FA-APMA-DMA-Dox (polymer 16) pre-incubated at pH 6.8 > APMA-DMA-Dox (polymer 15) pre-incubated at pH 6.8 > HPMA-Dox (polymer 13) > FAHPMA-Dox (polymer 14). As mentioned above, the inferior spheroids inhibiting ability of single-modified FA-HPMA (186% of original spheroid volume on 7th day) when compared with non-modified HPMA-Dox (130% of original spheroid volume on 7th day), could possibly be due to the insufficiently enhanced cell uptake mediated by folate-receptor mediation because of the “folate dilemma”. Although the flow cytometry results showed that internalization of folate-modified HPMA was slightly more efficient than nonmodified one at 2 h, both internalization rates still remained low compared with other conjugates. The insignificant active targeting exhibited by FA-HPMA-Dox (polymer 14) might become negligible especially when incubating for such a long time as 7 days. However, as the positive charge was introduced, the impact of folate on the spheroids growth inhibition started to reverse. The inhibitory effects of FA-APMA-DMA-Dox (78% of original spheroid volume on 7th day) and APMA-DMA-Dox (104% of original spheroid volume on 7th day) pre-incubated at pH 6.8 for 4 h were both stronger than non-charged conjugates due to significantly increased cell uptake (p < 0.05), while dual-modified FA-APMADMA-Dox (polymer 16) exhibited greatest reduction in spheroid size. Possibly the re-exposed cationic charge not only facilitated the cell uptake via electrostatic interaction, but also helped to overcome the shielding effect of HPMA chain and enhanced the binding efficiency of folate to its receptor. Therefore, the active targeting advantages of folate could be maximally exploited. Expectedly, the inhibitory effect of FA-APMA-DMA-Dox (polymer 16) on tumor spheroids also exhibited pH-dependent manner. The growth of spheroids treated with FA-APMA-DMA-Dox preincubated at pH 6.8 for 4 h was inhibited to a significantly greater extent than treated with the conjugates without pre-incubation (144% of original spheroid volume on 7th day, p < 0.05). Taken previous results together, the pH-selective inhibitory effect by FAAPMA-DMA-Dox (polymer 16) was believed to be attributed to the mild acidic pH-triggered hydrolysis of charge shielding group DMA, thus confirming that FA-APMA-DMA-Dox (polymer 16) could only further enhance the anticancer efficacy in response to the tumor extracellular pH. 4. Conclusion In the present study, in order to overcome the “folate dilemma” and maximally exploit the active targeting ability of folate, the positive charge was introduced into folate modified doxorubicin delivery system based on HPMA conjugates. The cell internalization mechanism study revealed that the positive charge not only facilitated the cell uptake via absorptive endocytosis, but also helped to overcome the shielding effect of HPMA chain and enhanced the binding efficiency of folate to its receptor due to close proximity of the conjugates to the cell surface by the electronic adhesion. To address the problem of non-specific binding by positive charge in the circulation, the charge shielding/deshielding approach was employed. The Dox-loaded, charge-reversible, folate-decorated HPMA conjugates were proved to be able to efficiently response to tumor extracellular pH to undergo a quick cationic charge regaining process and significantly enhanced its cell uptake by the combination of electrostatic absorptive endocytosis and receptormediated endocytosis in folate receptor positive Hela cells, which eventually led to enhanced anticancer activity after intracellular drug release triggered at pH 5 in endo/lysosomal compartment.

This multifunctional drug delivery system was demonstrated to be highly selective and effective in killing tumor cells, which provided a versatile approach for efficient cancer therapy.

Acknowledgments The research described above was supported by the National Natural Science Foundation of China (81072600).

References [1] Kope cek J, Kope cková P. HPMA copolymers: origins, early developments, present, and future. Adv Drug Deliv Rev 2010;62:122e49. [2] Lammers T. Improving the efficacy of combined modality anticancer therapy using HPMA copolymer-based nanomedicine formulations. Adv Drug Deliv Rev 2010;62:203e30.  [3] Ulbrich K, Subr V. Structural and chemical aspects of HPMA copolymers as drug carriers. Adv Drug Deliv Rev 2010;62:150e66. [4] Kope cek J, Kope cková P, Minko T, Lu Z-R. HPMA copolymereanticancer drug conjugates: design, activity, and mechanism of action. Eur J Pharm Biopharm 2000;50:61e81. [5] Lu ZR, Shiah JG, Sakuma S, Kope cková P, Kope cek J. Design of novel bioconjugates for targeted drug delivery. J Control Release 2002;78:165e73. [6] Tijerina M, Kope cková P, Kope cek J. Correlation of subcellular compartmentalization of HPMA copolymer-Mce6 conjugates with chemotherapeutic activity in human ovarian carcinoma cells. Pharm Res 2003;20:728e37. [7] Nori A, Jensen KD, Tijerina M, Kope cková P, Kopecek J. Tat-conjugated synthetic macromolecules facilitate cytoplasmic drug delivery to human ovarian carcinoma cells. Bioconjug Chem 2003;14:44e50. [8] Nori A, Jensen KD, Tijerina M, Kope cková P, Kope cek J. Subcellular trafficking of HPMA copolymereTat conjugates in human ovarian carcinoma cells. J Control Release 2003;91:53e9. [9] Yang Y, Zhou Z, He S, Fan T, Jin Y, Zhu X, et al. Treatment of prostate carcinoma with (galectin-3)-targeted HPMA copolymer-(G3-C12)-5-fluorouracil conjugates. Biomaterials 2012;33:2260e71. [10] Journo-Gershfeld G, Kapp D, Shamay Y, Kope cek J, David A. Hyaluronan oligomers-HPMA copolymer conjugates for targeting paclitaxel to CD44overexpressing ovarian carcinoma. Pharm Res 2012;29:1121e33. [11] Pike DB, Ghandehari H. HPMA copolymerecyclic RGD conjugates for tumor targeting. Adv Drug Deliv Rev 2010;62:167e83. [12] Lu Y, Low PS. Folate-mediated delivery of macromolecular anticancer therapeutic agents. Adv Drug Deliv Rev 2012;64:342e52. [13] Mornet E, Carmoy N, Lainé C, Lemiègre L, Le Gall T, Laurent I, et al. Folateequipped nanolipoplexes mediated efficient gene transfer into human epithelial cells. Int J Mol Sci 2013;14:1477e501. [14] Barz M, Canal F, Koynov K, Zentel R, Vicent MaJ. Synthesis and in vitro evaluation of defined HPMA folate conjugates: influence of aggregation on folate receptor (FR) mediated cellular uptake. Biomacromolecules 2010;11: 2274e82. [15] Canal F, Vicent MJ, Pasut G, Schiavon O. Relevance of folic acid/polymer ratio in targeted PEGeepirubicin conjugates. J Control Release 2010;146:388e99. [16] Valencia PM, Hanewich-Hollatz MH, Gao W, Karim F, Langer R, Karnik R, et al. Effects of ligands with different water solubilities on self-assembly and properties of targeted nanoparticles. Biomaterials 2011;32:6226e33. [17] McNeeley KM, Karathanasis E, Annapragada AV, Bellamkonda RV. Masking and triggered unmasking of targeting ligands on nanocarriers to improve drug delivery to brain tumors. Biomaterials 2009;30:3986e95. [18] McNeeley KM, Annapragada A, Bellamkonda RV. Decreased circulation time offsets increased efficacy of PEGylated nanocarriers targeting folate receptors of glioma. Nanotechnology 2007;18:385101. [19] Liu J, Bauer H, Callahan J, Kope cková P, Pan H, Kope cek J. Endocytic uptake of a large array of HPMA copolymers: elucidation into the dependence on the physicochemical characteristics. J Control Release 2010;143:71e9. [20] Duncan R, Izzo L. Dendrimer biocompatibility and toxicity. Adv Drug Deliv Rev 2005;57:2215e37. [21] Sui M, Liu W, Shen Y. Nuclear drug delivery for cancer chemotherapy. J Control Release 2011;155:227e36. [22] Hu J, Miura S, Na K, Bae YH. pH-responsive and charge shielded cationic micelle of poly (L-histidine)-block-short branched PEI for acidic cancer treatment. J Control Release 2013;172:69e76. [23] Jin E, Zhang B, Sun X, Zhou Z, Ma X, Sun Q, et al. Acid-active cell-penetrating peptides for in vivo tumor-targeted drug delivery. J Am Chem Soc 2013;135: 933e40. [24] Zhuang J, Chacko R, Amado Torres DF, Wang H, Thayumanavan S. Dual stimuliedual response nanoassemblies prepared from a simple homopolymer. ACS Macro Lett 2013;3:1e5. [25] Yuan YY, Mao CQ, Du XJ, Du JZ, Wang F, Wang J. Surface charge switchable nanoparticles based on zwitterionic polymer for enhanced drug delivery to tumor. Adv Mater 2012;24:5476e80.

L. Li et al. / Biomaterials 35 (2014) 5171e5187 [26] Oh NM, Kwag DS, Oh KT, Youn YS, Lee ES. Electrostatic charge conversion processes in engineered tumor-identifying polypeptides for targeted chemotherapy. Biomaterials 2012;33:1884e93. [27] Yoon SR, Yang HM, Park CW, Lim S, Chung BH, Kim JD. Charge-conversional poly (amino acid) s derivatives as a drug delivery carrier in response to the tumor environment. J Biomed Mater Res A 2012;100:2027e33. [28] Du JZ, Du XJ, Mao CQ, Wang J. Tailor-made dual pH-sensitive polymere doxorubicin nanoparticles for efficient anticancer drug delivery. J Am Chem Soc 2011;133:17560e3. [29] Cheng R, Meng F, Deng C, Klok HA, Zhong Z. Dual and multi-stimuli responsive polymeric nanoparticles for programmed site-specific drug delivery. Biomaterials 2013;34:3647e57. [30] Du JZ, Mao CQ, Yuan YY, Yang XZ, Wang J. Tumor extracellular acidity-activated nanoparticles as drug delivery systems for enhanced cancer therapy. Biotechnol Adv; 2013. http://dx.doi.org/10.1016/j.biotechadv.2013.08.002. [31] Omelyanenko V, Kopeckova P, Gentry C, Kopecek J. Targetable HPMA copolymer-adriamycin conjugates. Recognition, internalization, and subcellular fate. J Control Release 1998;53:25e37.  Ríhová   ák C, [32] Etrych T, Mrkvan T, Chytil P, Kon B, Ulbrich K. N-(2-hydroxypropyl) methacrylamide-based polymer conjugates with pH-controlled activation of doxorubicin. I. New synthesis, physicochemical characterization and preliminary biological evaluation. J Appl Polym Sci 2008;109:3050e61. [33] York AW, Zhang Y, Holley AC, Guo YL, Huang FQ, McCormick CL. Facile synthesis of multivalent folate-block copolymer conjugates via aqueous RAFT polymerization: targeted delivery of siRNA and subsequent gene suppression. Biomacromolecules 2009;10:936e43.  hová B, Ulbrich K. New HPMA copolymers containing [34] Etrych T, Jelınková M, Rı doxorubicin bound via pH-sensitive linkage: synthesis and preliminary in vitro and in vivo biological properties. J Control Release 2001;73:89e102. [35] Xiao J, Duan X, Yin Q, Zhang Z, Yu H, Li Y. Nanodiamonds-mediated doxorubicin nuclear delivery to inhibit lung metastasis of breast cancer. Biomaterials 2013;34:9648e56.

5187

[36] Duncan R, Cable HC, Rejmanova P, Kopecek J, Lloyd JB. Tyrosinamide residues enhance pinocytic capture of N-(2-hydroxypropyl)methacrylamide copolymers. Biochim Biophys Acta 1984;799:1e8. [37] Qu D, Lin H, Zhang N, Xue J, Zhang C. In vitro evaluation on novel modified chitosan for targeted antitumor drug delivery. Carbohydr Polym 2012;92: 545e54. [38] Moradi E, Vllasaliu D, Garnett M, Falcone F, Stolnik S. Ligand density and clustering effects on endocytosis of folate modified nanoparticles. RSC Adv 2012;2:3025e33. [39] Gabrielson NP, Pack DW. Efficient polyethylenimine-mediated gene delivery proceeds via a caveolar pathway in HeLa cells. J Control Release 2009;136:54e61. [40] Pelkmans L, Helenius A. Endocytosis via caveolae. Traffic 2002;3:311e20.  hová B. HPMA copolymers with [41] Ulbrich K, Etrych T, Chytil P, Jelınková M, Rı pH-controlled release of doxorubicin: in vitro cytotoxicity and in vivo antitumor activity. J Control Release 2003;87:33e47.  Sírová    ák C, [42] Chytil P, Etrych T, Kon M, Mrkvan T, Ríhová B, et al. Properties of HPMA copolymeredoxorubicin conjugates with pH-controlled activation: effect of polymer chain modification. J Control Release 2006;115:26e36. [43] Thomas JJ, Rekha M, Sharma CP. Unraveling the intracellular efficacy of dextran-histidine polycation as an efficient nonviral gene delivery system. Mol Pharm 2011;9:121e34.  [44] Ulbrich K, Subr Vr. Polymeric anticancer drugs with pH-controlled activation. Adv Drug Deliv Rev 2004;56:1023e50. [45] Minotti G, Menna P, Salvatorelli E, Cairo G, Gianni L. Anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity. Pharmacol Rev 2004;56:185e229. [46] Wang X, Zhen X, Wang J, Zhang J, Wu W, Jiang X. Doxorubicin delivery to 3D multicellular spheroids and tumors based on boronic acid-rich chitosan nanoparticles. Biomaterials 2013;34:4667e79. [47] Kim TH, Mount CW, Gombotz WR, Pun SH. The delivery of doxorubicin to 3-D multicellular spheroids and tumors in a murine xenograft model using tumorpenetrating triblock polymeric micelles. Biomaterials 2010;31:7386e97.

́

́

́