Bloodstream Stability Predetermines the Antitumor Efficacy of Micellar

Article Cite This: Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Bloodstream Stability Predetermines the Antitumor Efficacy of Micellar Polymer−Doxorubicin Drug Conjugates with pH-Triggered Drug Release Petr Chytil,†,‡ Milada Šírová,‡,§ Júlia Kudlácǒ vá,† Blanka Ř íhová,§ Karel Ulbrich,† and Tomás ̌ Etrych*,† †

Institute of Macromolecular Chemistry, Czech Academy of Sciences, Heyrovského náměstı ́ 2, 162 06 Prague 6, Czech Republic Institute of Microbiology, Czech Academy of Sciences, Vídeňská 1083, 142 20 Prague 4, Czech Republic

§

ABSTRACT: Herein, the biodegradable micelle-forming amphiphilic N-(2-hydroxypropyl) methacrylamide (HPMA)-based polymer conjugates with the anticancer drug doxorubicin (Dox) designed for enhanced tumor accumulation were investigated, and the influence of their stability in the bloodstream on biodistribution, namely, tumor uptake, and in vivo antitumor efficacy were evaluated in detail. Dox was attached to the polymer carrier by a hydrazone bond enabling pH-controlled drug release. While the polymer−drug conjugates were stable in a buffer at pH 7.4 (mimicking bloodstream environment), Dox was released in a buffer under mild acidic conditions modeling the tumor microenvironment or cells. The amphiphilic polymer carriers differed in the structure of hydrophobic cholesterol derivative moieties bound to the HPMA copolymers via a hydrolyzable hydrazone bond, exhibiting different rates of micellar structure disintegration at various pH values. Considerable dependence of the studied polymer−drug conjugate biodistribution on the stability of the micellar structure was observed in neutral, bloodstream-mimicking, environment, showing that a faster rate of the micelle disintegration in pH 7.4 increased the conjugate blood clearance, decreased tumor accumulation, and significantly reduced the tumor:blood and tumor:muscle ratios. Similarly, the final therapeutic outcome was strongly affected by the stability of the micellar structure because the most stable micellar conjugate showed an almost similar therapeutic outcome as the water-soluble, nondegradable, high-molecular-weight starlike HPMA copolymer−Dox conjugate, which was highly efficient in the treatment of solid tumors in mice. Based on the results, we conclude that the bloodstream stability of micellar polymer−anticancer drug conjugates, in addition to their low side toxicity, is a crucial parameter for their efficient solid tumor accumulation and high in vivo antitumor activity. KEYWORDS: polymer micelles, degradation, pH-controlled release, HPMA copolymer, solid tumor treatment

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INTRODUCTION The synthetic polymer carriers based on N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers and their drug conjugates represent very attractive agents suitable for specific drug delivery, thanks to their water solubility, biocompatibility, nontoxicity, and nonimmunogenicity.1,2 A typical application is the treatment of cancer and inflammatory diseases.2,3 The nanosized polymer carriers are accumulated in solid tumors through the generally accepted concept of the enhanced permeability and retention (EPR) effect.4,5 By conjugating lowmolecular-weight anticancer drugs, mainly cytostatic agents (e.g., doxorubicin (Dox), pirarubicin, or docetaxel), to HPMAbased copolymers, the tumor accumulation of the drugs can be significantly enhanced. Following enzymolytic or pH-triggered © XXXX American Chemical Society

controlled drug release from the accumulated conjugates provides a high concentration of active drugs in the tumor cells or tissue, resulting in a highly improved therapeutic response. After intravenous application, high-molecular-weight HPMA copolymer conjugates with a size or molecular weight (Mw) exceeding the renal filtration limit (approximately 50000 g/mol Special Issue: New Directions for Drug Delivery in Cancer Therapy Received: Revised: Accepted: Published: A

February 12, 2018 March 9, 2018 March 15, 2018 March 15, 2018 DOI: 10.1021/acs.molpharmaceut.8b00156 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Article

Molecular Pharmaceutics

believe that a similar correlation between the biodegradability rate of the carrier and final therapeutic outcome is crucial to understand the micellar nanomedicine functioning in the tumor treatment. The principal aim of this study was to recognize the relationship between degradation of the polymer micellar structures, e.g., the hydrolysis rate of spacers linking the hydrophilic and hydrophobic parts of the amphiphilic HPMA copolymer−Dox conjugates leading to the disintegration of high-molecular-weight micellar structure, and in vivo anticancer activity. We focused our study on the biodistribution, tumor accumulation, and anticancer activity of the biodegradable micelle-forming polymer−Dox conjugates with respect to the detailed polymer carrier structure.

and 10 nm in diameter) persist in the circulation at significant concentrations for much longer than the free drug, i.e., for more than a week.6 The extent of passive accumulation of macromolecules in solid tumors strongly depends upon the increasing size of polymer coils in aqueous solutions, but this trend reverses at very high molecular weights.7−9 It was found that this effect is molecular-weight dependent; blood clearance rate decreases with increasing Mw (up to a limit of approximately 500000 g/mol for HPMA copolymer conjugates10). Thus, to enhance the tumor accumulation of polymer−drug conjugates, their size should be increased above the renal threshold to reach longer circulation and efficient time-dependent tumor accumulation. However, such polymer conjugates should be biodegradable to prevent longterm accumulation in the body after fulfilling their role as a drug carrier. Various high-molecular-weight HPMA copolymer carriers designed for controlled degradation, resulting in short polymer fragments and enabling fast elimination from the body by renal filtration, were successfully synthesized and tested.10,11 Another approach to achieve long circulating polymer carrier systems with controlled degradation involves the self-assembly of amphiphilic HPMA copolymers that form sufficiently stable micellar structures with a size exceeding the limit of the renal threshold.12 A relatively effortless way to prepare micelle-forming amphiphilic HPMA copolymers resided in the introduction of a small amount of hydrophobic cholesterol moieties in the copolymer carrier structure. The amphiphilic polymer conjugate with Dox bound by pH-sensitive hydrazone bond forming micelles in aqueous solutions showed a remarkable therapeutic effect compared with the effect of the hydrophilic polymer conjugate completely dissolved (in the form of unimolecular polymer coils) in the solutions.13,14 Afterward, similar amphiphilic polymer−Dox conjugates containing pHsensitive hydrazone bond in spacers between the hydrophilic polymer carrier and hydrophobic cholesterol moieties were synthesized and their physicochemical properties, especially self-assembly and micelle formation in aqueous solutions and their pH-controlled disintegration facilitating the carrier elimination from the body, were studied in detail.15,16 A very important issue still missing in the literature is the optimization of the micelle disintegration controlled by the rate of the amphiphilic polymer biodegradation. This is in strong agreement with recent papers alerting the low delivery efficiency of nanoparticles, which should be studied collectively with respect to the physicochemical parameters of nanoparticles, tumor models, and cancer types.17 The potential causes of the poor delivery efficiency should be studied also from the perspectives of tumor biology (intercellular versus transcellular transport, the EPR effect, and physicochemicaldependent transport of nanoparticles through the tumor stroma) as well as competing organs (mononuclear phagocytic and renal systems).18 Recently, we determined that the circulation time of the active agent was closely related to the desired biological outcome using in vivo noninvasive multispectral optical imaging of dual fluorescently labeled HPMA copolymer.19 Too fast release of the drug model, i.e., far red dye, from the polymer carrier at neutral (blood) pH resulted only in a minor accumulation of the drug model in tumor tissue due to its faster blood clearance. However, the presence of a more stable spacer in the HPMA copolymer conjugate structure enabled a prolonged circulation time in blood and, thus, enhanced tumor accumulation of the drug model. We

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MATERIALS AND METHODS Chemicals. We purchased 1-aminopropan-2-ol, methacryloyl chloride, 6-aminohexanoic acid, methyl 6-aminohexanoate hydrochloride, hydrazine hydrate, tert-butyl carbazate, trifluoroacetic acid (TFA), N,N′-dicyclohexylcarbodiimide, 4(dimethylamino)pyridine, cholesterol, cholest-4-en-3-one, 5αcholestanone, dicyclohexylcarbodiimide, dimethyl sulfoxide (DMSO), N,N-diisopropylethylamine, 2,2′-azoisobutyronitrile, and 4,4′-azobis(4-cyanopentanoic acid) from Sigma-Aldrich. We purchased 4-(2-oxopropyl)benzoic acid from Rieke Metals. Poly(amido amine) (PAMAM) dendrimer (G2, amine surface) was purchased from Andrews ChemServices. All other chemicals and solvents were of analytical grade. The solvents were dried and purified by conventional procedures. Synthesis of Monomers and Cholesterol Derivatives. HPMA was prepared as described in ref 20, 6-methacrylamidohexanohydrazide (MA-Ahx-NHNH2) was prepared as described in ref 21, 1-(tert-butoxycarbonyl)-2-(6-methacrylamido hexanoyl)hydrazine (MA-Ahx-NHNH-Boc) was synthesized as described in ref 22, and cholest-5-en-3β-yl 4-(2-oxopropyl)benzoate (Opb-Chol) was prepared as previously described in ref 16. The structure and purity of the monomers and cholesterol derivative were examined by 1H NMR (Bruker spectrometer, 300 MHz) and HPLC (Shimadzu 20VP) using a C18 reversephase Chromolith Performance RP-18e (4.6 × 100 mm) column with photo diode array detection (Shimadzu SPDM20A). The eluent was water−acetonitrile with a gradient of 5−95 vol % acetonitrile, 0.1% TFA, and a flow rate of 5 mL/ min. Synthesis of Polymer Precursors. The amphiphilic statistical copolymers bearing either cholest-4-en-3-one (P1), Opb-Chol (P2), or 5α-cholestanone (P3) were synthesized according to our previous paper using the statistical copolymer poly(HPMA-co-MA-Ahx-NHNH2) prepared by free radical polymerization of HPMA and the monomer containing a hydrazide group (MA-Ahx-NHNH2) in methanol initiated by 2,2′-azoisobutyronitrile, followed by conjugation with keto derivatives of cholesterol (theoretical content in the reaction mixture: 2 mol % of cholest-4-en-3-one or 5α-cholestanone; 1.5 mol % of Opb-Chol), forming a hydrazone linkage.16 The star polymer precursor (SP) was synthesized by grafting the semitelechelic HPMA copolymer poly(HPMA-co-MA-AhxNHNH-Boc)-TT with the terminal thiazolidine-2-thione (TT) chain end group onto the second generation PAMAM dendrimer containing terminal amino groups followed by hydrazide group deprotection as described in ref 23. The semitelechelic copolymer was prepared by free radical polymerB

DOI: 10.1021/acs.molpharmaceut.8b00156 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics Table 1. Characteristics of the HPMA Copolymer−Dox Conjugates

a

conjugate no.

structure

hydrophobic moiety

Mwa (g/mol)

Đa

drug content (wt %)

RH (nm)

P1−Dox P2−Dox P3−Dox SP−Dox

micellar micellar micellar starlike

cholest-4-en-3-one Opb-Chol 5α-cholestanone

25500 38000 24500 250000

1.8 1.8 1.9 1.9

8.1 9.4 8.2 9.5

14.8 12.5 13.0 12.6

Determined in the methanol/acetic buffer (0.3 M, pH 6.5) mobile phase (4:1).

activity of the polymer conjugates: EL4.IL-2 subline of EL4 Tcell lymphoma (ATCC TIB-181), mammary carcinoma 4T1 (ATCC CRL-2539), and colon adenocarcinoma CT26 (ATCC CRL-2638). The cell lines were maintained as recommended by the provider. Murine head and neck carcinoma SCC727 was kindly donated by prof. D. M. Lathers (USA), and maintained in RPMI 1640, supplemented with 2 mM L-glutamine and fetal calf serum (FCS). The media and supplements were obtained from Sigma-Aldrich, and FCS (10% for all cell lines) was obtained from Invitrogen. Instead of EL4 T-cell lymphoma, its EL4.IL-2 subline was explored in 3H-thymidine incorporation assays because the EL4 cells do not incorporate thymidine at a sufficient intensity. The cells were cultivated in 96-well flat-bottomed (FB) culture plates (Thermo Scientific) with various concentrations of polymer−Dox conjugates or free Dox·HCl as a positive control. The cell counts at the beginning of cultivation were 5,000/well or 2,500/well in experiments with 4T1 cells. The cell growth inhibition was calculated as the IC50, the concentration of cytotoxic drug that inhibits the proliferation by 50%. At least four parallel samples were used for each experimental condition. In Vivo Tumor Treatment. Inbred C57BL/6 (H-2b) female mice were obtained from the Institute of Physiology, Czech Academy of Sciences, Prague, Czech Republic. EL4 Tcell lymphoma (ATCC TIB-39) was used as the in vivo model of the solid tumor. The mice (two to three months old, weighing approximately 23 g) were subcutaneously (sc) transplanted at day zero with 1 × 105 EL4 cells and were treated with the polymer conjugates injected intravenously (iv) at day 8, when the diameter of the tumor focuses was 6−8 mm. The mice were injected with a single dose of the polymer−Dox conjugate, the equivalent of 7.5 mg Dox/kg. Tumor growth, the body weight as a parameter of overall toxicity, and survival time were monitored. The tumor volume was calculated as V = a·b2/ 2, where a = longer diameter, and b = smaller diameter. To document the contribution of the immune system to the therapy, the long-term survivors (LTS) in the EL4 lymphoma model were challenged with the same dose of EL4 lymphoma cells (1 × 105) injected sc at day 120 after the first tumor transplantation and were left untreated. The tumor growth and survival were monitored. In all animal work, institutional guidelines for the care and use of laboratory animals were strictly followed under a protocol approved by the Institutional Animal Care and Use Committee of Czech Academy of Sciences, and the experiments were conducted in compliance with local and European guidelines. Dox Accumulation in the Tumor. The EL4 tumorbearing mice were administered iv with polymer−Dox conjugates, a single dose of 10 mg Dox equiv/kg. At specified time intervals, the mice were sacrificed. Heparinized blood and samples of tumor, skeletal muscle, and liver were harvested. The weighed tissue was homogenized in 1 mL of PBS using a

ization of HPMA and MA-Ahx-NHNH-Boc in DMSO initiated with TT-functionalized azo-initiator 4,4′-azobis(4-cyanopentanoic acid)24 as described previously.25 Synthesis of Polymer Conjugates with Doxorubicin. Polymer−drug conjugates P1−Dox, P2−Dox, P3−Dox, and SP−Dox were synthesized by reaction of the respective polymer precursors bearing hydrazide groups P1−P3 or SP with Dox·HCl in methanol in the dark, as described previously.26

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METHODS The content of the cholesterol-derived moieties in the copolymers was determined by HPLC analysis after acid hydrolysis in 6 M HCl followed by extraction into chloroform as described previously.16 The drug content in the polymer−drug conjugates (Table 1) was determined spectrophotometrically, and the absence of unbound drug in the polymer−drug conjugates was verified by HPLC. The molecular weights (Mw) and dispersity (Đ) of the polymer precursors and their drug conjugates were determined by HPLC equipped with a GPC column (TSKgel Super SW3000, 300 × 4.6 mm; 4 μm) and the photodiode array, differential refractive index Optilab-rEX, and multiangle light scattering DAWN HELEOS II (Wyatt Technology Co., USA) detectors using a methanol−sodium acetate buffer (0.3 M; pH 6.5) mixture (80:20 vol %; flow rate 0.3 mL/min). The Mw and Đ were calculated using ASTRA VI software, and a refractive increment index dn/dc 0.175 mL/g was used. The Optilab-rEX detector enables direct determination of refractive increment (dn/dc) of the polymers, and the solvent refractive index provides 100% recovery of the injected sample from the column. The hydrodynamic radii (RH) of the polymer micelles in phosphate-buffered saline (0.01 g/mL; pH 7.4) were measured using a Nano-ZS instrument (ZEN3600, Malvern). The intensity of the scattered light was detected at angle θ = 173° using a laser with a wavelength of 632.8 nm. To evaluate the dynamic light scattering data, the DTS(Nano) program was used. The values were a mean of at least five independent measurements. Values were not extrapolated to zero concentration. In Vitro Drug Release from the Polymer−Dox Conjugates. The rates of Dox release were investigated in phosphate buffers at pH 5.5, 6.0, 6.5, or 7.4 (0.1 M, with 0.05 M NaCl) at 37 °C using a polymer concentration of 5 mg/mL. The amount of released Dox was determined by HPLC using a GPC column (described above) from the relative area of peaks (UV/vis detection at 488 nm) corresponding to the released drug and polymer-bound Dox. The values were expressed as the means of three independent experiments. In Vitro Toxicity of the Polymer−Dox Conjugates. The following murine tumor cancer cell lines were purchased from the ATCC and used for the in vitro evaluation of the cytotoxic C


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Figure 1. Schematic structure of amphiphilic polymer−Dox conjugates P1−Dox to P3−Dox (A) and SP−Dox (B). Release of cholesterol moieties from copolymers P1−P3 at pH 7.4 and 37 °C, mimicking the bloodstream environment (C).

glass homogenizer. The homogenates that were free of coarse tissue remnants as well as blood were frozen at −20 °C until sample processing. Three mice were used per conjugate/time interval. The samples were tested for the total content of Dox, i.e., both free and polymer-bound Dox. Dox content determination was performed after quantitative acid hydrolysis in 1 M HCl at 50 °C. After incubation for 1 h, doxorubicinone (aglycon of Dox) was extracted with chloroform, the organic phase was evaporated to dryness, and the remaining solid was completely dissolved in methanol. The aglycon was analyzed using a gradient-based HPLC Shimadzu system equipped with a fluorescence detector (Shimadzu RF-10Axl) (λexc = 488 nm, λem = 560 nm) and reversed-phase column (Chromolith High Resolution RP-18e, 4.6 × 100 mm). The eluent was water− acetonitrile with a gradient of 5−95 vol % acetonitrile, 0.1% TFA, and a flow rate of 5 mL/min. A calibration curve was obtained by the injection of exact amounts of free Dox·HCl into the blood and tumor tissue homogenates obtained from untreated animals. The calibration samples were analyzed after homogenization, hydrolysis, and extraction. All experiments were carried out in quadruplicate. Statistical Analysis. Analysis of significance was conducted using Student’s t test, comparison of the survival times was conducted using the log-rank (Mantel−Cox) test, and statistical

analysis was performed with GraphPad Prism software. Significance was set as p < 0.05.

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RESULTS AND DISCUSSION Here, we present the results of the biological evaluation of the biodegradable micelle-forming HPMA copolymer conjugates with anticancer agent Dox bound to the polymer via the stimulus-sensitive spacer. The conjugates differ in the rate of micellar drug carrier disintegration, which can potentially influence the drug pharmacokinetics and elimination of the polymer carrier system from the body. Physicochemical properties and solution behavior of the micelle-forming polymer−Dox conjugates were discussed in detail previously.15,16 We proved that the rate of micelle disintegration under conditions simulating the bloodstream strongly corresponded with the structure of the biodegradable hydrazone spacer between the hydrophilic HPMA copolymer carrier and hydrophobic cholesterol moieties. Here, we focused mainly on detailed biological evaluation, e.g., biodistribution study, tumor accumulation, and anticancer activity, of the micelle-forming polymer−Dox conjugates containing cholesterol moieties bound to polymer via spacers differing in hydrolysis rates in bloodlike conditions. The slowest release rate was obtained in the case when cholest-4-en-3-one was used in the polymer− drug conjugate P1−Dox, and the fastest release rate was D


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Figure 2. Release of Dox from polymer conjugates in phosphate buffers at pH 7.4, 6.5, 6.0, and 5.5 at 37 °C. P1−Dox, ◆; P2−Dox, □; P3−Dox, ▲; SP−Dox, ●, dotted line.

lytic release as documented many times previously, e.g., for Dox, pirarubicin, docetaxel, and dexamethasone.28 The mechanism of action of the polymer−drug conjugates enabling the pH-controlled drug release in tumor tissue or cells has been discussed in detail in the literature.29 Here, we also observed a strong pH-dependent rate of Dox release at physiological 37 °C, i.e., the release rate was elevated with decreasing pH (see Figure 2). While only a minor release of Dox was determined (up to 10% of released drug within 24 h) at blood pH (pH 7.4), even a slight pH decrease to 6.5, i.e., pH in extracellular tumor tissue (typically 6.5−6.8), resulted in a significant increase in drug release (approximately 30% within 24 h). A further pH decrease to 6.0 or 5.5, which mimics conditions in endosomes or lysosomes of tumor cells, led to approximately 55% or 75% of released Dox within 24 h, respectively. As expected, there were no significant differences in the drug release rates from particular conjugates based on either amphiphilic or fully hydrophilic starlike copolymers because Dox is randomly distributed throughout the hydrophilic chains with the same solvation in all conjugates. Similar results were obtained previously.14,16 We proved that micelle-forming polymer−Dox conjugates fulfill the basic criteria for an efficient anticancer prodrug, i.e., stability during blood transportation, exemplified by the incubation of polymer conjugates in a buffer at neutral pH, and fast release of the active drug after entering tumor tissue or tumor cells, simulated by the incubation of the conjugates in various acidic buffers. In Vitro Cytotoxicity of Polymer−Dox Conjugates. The cytotoxic activity of the polymer conjugates was tested as an in vitro proof of activity of the conjugates using two Dox-sensitive cell lines (EL.4.IL-2 and 4T1), and two other less Dox-sensitive

obtained when 5α-cholestanone was used in the conjugate P3− Dox. As a positive control, the highly therapeutically potent nondegradable, water-soluble, high-molecular-weight starlike HPMA copolymer−Dox conjugate (SP−Dox) was employed.10 The structures are shown in Figures 1A and 1B. In our previous work,16 we determined that only 2% of cholest-4-en-3-one was released from copolymer P1 within 5 days at neutral pH at physiological temperature. A faster release (9% within 5 days) and much faster release (37% within 5 days) were determined for OPB-Chol from P2 or for 5αcholestanone from P3 at pH 7.4 at 37 °C, respectively (see Figure 1). The differences in the hydrolysis rates can be explained by the stabilization of the hydrazone bond due to the presence of the double bond (P1) or an aromatic ring (P2) close to the hydrazone bond (see Figure 1C). Notably, all three micellar copolymers showed faster disintegration at acidic pH; thus, fast biodegradation to hydrophilic polymers eliminable from the organism should occur after the accumulation into tumor tissue. The disassembly of the micellar polymer carrier is also influenced by the critical micellar concentration (cmc). Below cmc, unimerssingle polymer chainscannot form micelles and thus their blood clearance is faster and the tumor uptake lower when compared with the micellar carriers. This concentration was found very low for all studied polymers P1−P3 and moreover almost the same (about 3−4 mg/L).16 Thus, we presume that cmc values will not significantly influence the differences in pharmacokinetics of the studied micellar polymer conjugates. In Vitro Release of Dox from the Polymer−Drug Conjugates. Anticancer drugs bound to HPMA copolymer carriers by the hydrazone bond undergo pH-triggered hydroE


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Molecular Pharmaceutics Table 2. Cytotoxic Activitya of the Polymer Conjugates and Free Doxorubicin in Vitro EL4.IL-2

a

SCC7

4T1

CT26

conjugate no.

meanb

SD

meanb

SD

meanb

SD

meanb

SD

P1−Dox P2−Dox P3−Dox SP−Dox Dox·HCl

57.7 56.2 64.2 77.6 7.5

9.8 15.2 17.3 5.4 0.8

113.2 147.2 119.7 129.7 24.4

0.64 16.48 5.37 16.76 4.38

33.6 50.5 34.7 36.8 9.4

3.25 4.53 2.97 1.41 1.06

54.0 85.8 60.8 80.6 22.2

4.88 10.89 5.87 37.97 6.43

The cytotoxic activity was expressed as the IC50 value in ng Dox equiv/mL. bThe data from at least three independent experiments are included.

and eliminated, preventing its delivery to the tumor at later time points. On the other hand, the more stable carriers P2 and P1 mediated the tumor delivery comparably to the SP−Dox conjugate. Considering all the data from the biodistribution study, we can summarize that the stability of micellar carrier system in the blood environment plays a crucial role in its pharmacokinetic profile, being superior for the carrier system being more stable during the circulation in blood. The modest increase in blood clearance and decrease in tumor accumulation of the most stable micellar conjugate P1−Dox compared with SP−Dox could be ascribed to the relatively short and reversible accumulation of the micellar conjugate in the liver. Tumor:blood and tumor:muscle ratio analysis unambiguously showed the strong therapeutic potential of the degradable micellar polymer conjugate P1−Dox because the parameters were even better than those obtained for SP−Dox. The results of our studies show that the micellar conjugate P1−Dox is a good candidate for further detailed biological evaluation as a potential nanomedicine suitable for the treatment of cancer. Tumor Treatment. The treatment scheme in the EL4 Tcell lymphoma model bearing mice was set according to the former data determined during treatment with SP−Dox, a water-soluble, high-molecular-weight long-circulating polymer− Dox conjugate with a nondegradable carrier structure, which confers the conjugate ability to mediate high tumor accumulation of drug and excellent therapeutic effect. To compare the treatment potential of micellar polymer carrier systems with SP−Dox, considered an internal “gold standard” in our hands,10 the EL4 T-cell lymphoma-bearing mice were treated with a suboptimal single dose of micellar polymer conjugates P1−Dox to P3−Dox, and their effect was compared with that induced by the polymer conjugate SP−Dox, showing the same hydrodynamic size in solution (RH approximately 12− 13 nm). The mean tumor size after the treatment was virtually the same for conjugates P1−Dox and P2−Dox, being larger than that mediated by SP−Dox (Figure 4A). However, the tumor growth was only moderately retarded after the treatment with conjugate P3−Dox. In accordance with this trend, the survival of mice was the highest in the treatment with SP−Dox and decreased with the stability of the micellar carrier structure as described above. SP−Dox induced complete tumor regression and 62.5% of LTS, P1−Dox treatment resulted in 37.5% LTS, and P2−Dox treatment resulted in 12.5% LTS. The conjugate with the most labile structure (P3−Dox) prolonged the survival time but did not induce any complete tumor regression and tumor-free survival (Figure 4B). We assume that the difference in the carrier stability between P1−Dox and P2−Dox was not large enough to induce detectable difference in the tumor growth (Figure 4A), despite the fact that differences in blood clearance and tumor

cell lines, SCC7 and CT26. The cytotoxicity of the polymer micellar conjugates and SP−Dox was similar in all the tested cell lines (Table 2). Obviously, the in vitro cytotoxic activity of the polymer conjugates was not influenced by the stability of the polymer carrier systems, suggesting that the cytotoxic activity of the conjugates could be related rather to the Dox release from the polymer carrier. Thus, because the Dox release was virtually identical among all the tested polymer conjugates, the similarity of the IC50 values is not surprising. Dox Blood Clearance and Tumor Accumulation. As expected, when detecting Dox administered iv in the form of the conjugate with nondegradable carrier structure, Dox persisted in the blood for a longer time than the micellar conjugates P2−Dox and P3−Dox (Figure 3A). The blood clearance of the conjugate P1−Dox was virtually identical to that of SP−Dox, indicating significant stability of the micellar structure in peripheral blood within several days. The most labile micellar conjugate P3−Dox was removed from the circulation fairly quickly, thus proving the strong dependence of blood clearance on the carrier stability in the blood environment. Similarly, tumor-specific accumulation of Dox was dependent on the carrier stability, being most prominent for SP−Dox and P1−Dox. The Dox content was significantly lower in tumors in the case of less-stable conjugate P2−Dox and least-stable conjugate P3−Dox, ensuring about one-third of the Dox content in the tumor compared with the SP−Dox and P1−Dox conjugates at any time interval tested (Figure 3B). The accumulation of all the conjugates in skeletal muscles (Figure 3C) was low, again the highest for the P1−Dox conjugate, and the lowest (about half of the Dox content) for the P3−Dox conjugate, strongly corresponding to the persistence of the conjugates in blood. However, the accumulation of the most stable micellar conjugate P1−Dox in the liver (Figure 3D) was increased, being even higher than that detected when the SP−Dox conjugate was used. This indicates a more significant uptake of the micellar conjugate in liver tissue, a trend that has been often documented for various other micellar drug delivery systems. Importantly, the Dox content in the liver dropped within 48 h to reasonably low levels; at that time, the Dox content in the liver was comparable for all the used conjugates. The calculated value of the tumor:blood ratio (Figure 3E) was continuously raised between 12 and 96 h for all the conjugates tested, illustrating that Dox effectively accumulates in the tumor tissue. As expected, the highest value of the tumor:blood ratio was observed for the nondegradable conjugate SP−Dox and the conjugate with the most stable carrier structure P1−Dox. However, the calculated tumor:muscle ratio (Figure 3F) showed that the tumor accumulation of Dox was rather low when the conjugate P3−Dox was used, obviously because the polymer carrier was degraded too fast F


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Figure 3. Blood clearance and Dox accumulation in the tumors, skeletal muscle, and liver tested in EL4 T-cell lymphoma-bearing mice treated with micellar conjugates. C57BL/6 mice were injected sc with 1 × 105 EL4 lymphoma cells and were treated at day 8 with a single dose of the polymer conjugates. The mice were treated with 10 mg Dox equiv/kg injected iv at day 8. At the predetermined time intervals (12, 24, 48, and 96 h post treatment), the mice were sacrificed, and heparinized blood and tissue samples were collected for Dox content determination. Blood clearance at 6, 12, 24, 48, and 96 h following treatment (A); Dox accumulation in tumors (B); skeletal muscle (C); liver (D); Dox content calculated as the tumor:blood ratio (E); tumor:muscle ratio (F). Number of animals per condition, n = 3. Figure legends: P1−Dox, ◆; P2−Dox, □; P3−Dox,▼; SP−Dox,●, dotted line.

which is tumor specific and mainly mediated by CD8+ cytotoxic T cells, has been already documented in treatment with various HPMA copolymer conjugates carrying Dox or taxanes (docetaxel or paclitaxel) and in several murine models.30−33 The treatment-dependent development of the antitumor immune responses is considered one of the most important characteristics of the treatment using the HPMA copolymer drug carriers, suggesting the involvement of the immunogenic

accumulation of Dox were observed (Figure 3). Also, the difference in the survival time was statistically insignificant between P1−Dox and P2−Dox treatment (Figure 4B), but the trend toward better survival in the treatment using the more stable polymer carrier (P2−Dox) is clear. Notably, upon reinjection of the LTS with the same (i.e., lethal) dose of the tumor cells and without any other treatment, most of the LTS did not develop a second tumor, thus proving complete tumor resistance (Figure 4C). The tumor resistance, G


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together with reasonable RH of the conjugate, the right combination of properties for such a stimulus-responsive nanosystem.2 However, the nondegradability of the carrier structure poses a strong limit regarding a potential clinical use because the carrier cannot be simply removed from the body. The kidney filtration limit is assumed to lie between Mw = 50,000 and 70,000 g/mol depending on the structure of the HPMA−copolymer conjugate, while Mw of the SP−Dox conjugates is much higher. Thus, the micellar conjugates with a similar RH as the conjugate SP−Dox could potentially retain the benefits of the SP−Dox conjugate while reducing the limitation of the nondegradable carrier structure. Based on the results of the in vivo study, we can conclude that the most stable micellar conjugate P1−Dox seems to be the best candidate combining the enhanced antitumor efficacy together with biodegradability of the carrier structure and its safe elimination from the body. By comparison of results of experiments with SP−Dox and P1−Dox we can prove that an ideal biodegradable HPMAbased polymer system should be highly stable in circulation and simultaneously not show any significant liver uptake. The biodegradation should occur after the delivery into the tumor as a consequence of the mission fulfillment.

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CONCLUSION In the present study, we evaluated the influence of the structural aspects of amphiphilic HPMA copolymer−Dox conjugates forming micelles in aqueous solution on their biological effects in vivo. In detail, the impact of the stability of micelles in the bloodstream-mimicking environment on the cytotoxicity and mainly on biodistribution, i.e., blood clearance, tumor accumulation, liver, and muscle uptake, and antitumor efficacy against EL4 T-cell lymphoma, was determined. We showed that the stability of the micellar carrier system in the blood environment plays a major role in the pharmacokinetic profile, i.e., prolonged blood circulation and enhanced tumor accumulation of the drug. Polymer conjugates P2−Dox or P3− Dox forming biodegradable micelles (releasing 9 or 37% of cholesterol moiety within 5 days at pH 7.4, 37 °C, respectively) exhibited much faster blood clearance and lower tumor uptake and thus lower anticancer activity than the fully water-soluble starlike polymer conjugate SP−Dox with the same RH. By contrast, the most stable micelle-forming conjugate P1−Dox (releasing only approximately 2% of cholest-4-en-3-one within 5 days at pH 7.4) exhibited a similar pharmacokinetic profile. It was only affected by a short reversible increase in liver accumulation, which could be ascribed to the hydrophobic interaction of the amphiphilic polymer carrier with the liver tissue. It resulted in a slightly increased blood clearance and decreased tumor accumulation, leading to a slightly lower therapeutic outcome during the in vivo therapy. Finally, we can conclude that the micellar polymer−drug conjugates with similar RH as the water-soluble nondegradable starlike polymer−drug conjugate can potentially maintain the benefits of the highly efficient star conjugate while significantly reducing the limitation of the nondegradable polymer carrier structure. Based on the results, the most stable micellar conjugate P1−Dox with cholest-4-en-3-one as the cholesterol moiety seems to be the best candidate combining the enhanced antitumor efficacy together with the hydrolytic biodegradability of the carrier structure in the final tumor destination. The precisely defined structure, controlled hydrodynamic size, drug release, and degradation profiles of the micellar conjugate P1−

Figure 4. Tumor growth, survival, and tumor resistance in the treatment of murine EL4 T-cell lymphoma with micellar Dox conjugates. C57BL/6 mice were injected sc with 1 × 105 EL4 T-cell lymphoma cells and were treated at day 8 with a single dose of the polymer conjugates, 7.5 mg Dox equiv/kg administered iv. SP−Dox was used for comparison. The tumor growth (A), survival time (B), and tumor resistance of LTS after tumor cell retransplantation (C) were monitored. The data represent the average of two independent experiments, eight animals per group/experiment. The tumor growth is expressed as percent of the initial tumor size at day 8. Significance in the tumor size evaluated on day 28 is depicted in panel A, p < 0.05 and p < 0.01. All LTS from the two experiments were retransplanted (C): P1−Dox-cured, n = 6; P2−Dox-cured, n = 1, SP−Dox-cured, n = 10. New untreated control, n = 16. Untreated controls, ○; P1−Dox, ◆; P2−Dox, □; P3−Dox,▼; SP−Dox, ●, dotted line.

cancer cell death induced by the conjugates carrying drugs bound via a hydrazone bond.34 The starlike HPMA-based copolymer SP−Dox carrying Dox bound via the pH-sensitive bond is a highly effective drug delivery system with fair stability in the circulation and controlled drug delivery in the target tissue, which forms, H


Article


(9) Grayson, S. M.; Godbey, W. T. The role of macromolecular architecture in passively targeted polymeric carriers for drug and gene delivery. J. Drug Targeting 2008, 16 (5), 329−356. (10) Etrych, T.; Kovár,̌ L.; Strohalm, J.; Chytil, P.; Ř íhová, B.; Ulbrich, K. Biodegradable star HPMA polymer-drug conjugates: Biodegradability, distribution and anti-tumor efficacy. J. Controlled Release 2011, 154 (3), 241−248. (11) Pan, H. Z.; Sima, M.; Yang, J. Y.; Kopeček, J. Synthesis of LongCirculating, Backbone Degradable HPMA CopolymerDoxorubicin Conjugates and Evaluation of Molecular-Weight-Dependent Antitumor Efficacy. Macromol. Biosci. 2013, 13 (2), 155−160. (12) Talelli, M.; Rijcken, C. J. F.; van Nostrum, C. F.; Storm, G.; Hennink, W. E. Micelles based on HPMA copolymers. Adv. Drug Delivery Rev. 2010, 62 (2), 231−239. (13) Filippov, S. K.; Chytil, P.; Konarev, P. V.; Dyakonova, M.; Papadakis, C. M.; Zhigunov, A.; Pleštil, J.; Štěpánek, P.; Etrych, T.; Ulbrich, K.; Svergun, D. I. Macromolecular HPMA-Based Nanoparticles with Cholesterol for Solid-Tumor Targeting: Detailed Study of the Inner Structure of a Highly Efficient Drug Delivery System. Biomacromolecules 2012, 13 (8), 2594−2604. (14) Chytil, P.; Etrych, T.; Koňaḱ , Č .; Šírová, M.; Mrkvan, T.; Bouček, J.; Ř íhová, B.; Ulbrich, K. New HPMA copolymer-based drug carriers with covalently bound hydrophobic substituents for solid tumour targeting. J. Controlled Release 2008, 127 (2), 121−130. (15) Filippov, S. K.; Franklin, J. M.; Konarev, P. V.; Chytil, P.; Etrych, T.; Bogomolova, A.; Dyakonova, M.; Papadakis, C. M.; Radulescu, A.; Ulbrich, K.; Štěpánek, P.; Svergun, D. I. Hydrolytically Degradable Polymer Micelles for Drug Delivery: A SAXS/SANS Kinetic Study. Biomacromolecules 2013, 14 (11), 4061−4070. (16) Chytil, P.; Etrych, T.; Kostka, L.; Ulbrich, K. Hydrolytically Degradable Polymer Micelles for Anticancer Drug Delivery to Solid Tumors. Macromol. Chem. Phys. 2012, 213 (8), 858−867. (17) Wilhelm, S.; Tavares, A. J.; Dai, Q.; Ohta, S.; Audet, J.; Dvorak, H. F.; Chan, W. C. W. Analysis of nanoparticle delivery to tumours. Nat. Rev. Mater. 2016, 1 (5), 16014. (18) Sun, Q. H.; Zhou, Z. X.; Qiu, N. S.; Shen, Y. Q. Rational Design of Cancer Nanomedicine: Nanoproperty Integration and Synchronization. Adv. Mater. 2017, 29 (14), 1606628. (19) Chytil, P.; Hoffmann, S.; Schindler, L.; Kostka, L.; Ulbrich, K.; Caysa, H.; Mueller, T.; Mäder, K.; Etrych, T. Dual fluorescent HPMA copolymers for passive tumor targeting with pH- sensitive drug release II: Impact of release rate on biodistribution. J. Controlled Release 2013, 172 (2), 504−512. (20) Chytil, P.; Etrych, T.; Kříž, J.; Šubr, V.; Ulbrich, K. N-(2Hydroxypropyl)methacrylamide-based polymer conjugates with pHcontrolled activation of doxorubicin for cell-specific or passive tumour targeting. Synthesis by RAFT polymerisation and physicochemical characterisation. Eur. J. Pharm. Sci. 2010, 41 (3−4), 473−482. (21) Etrych, T.; Mrkvan, T.; Chytil, P.; Koňaḱ , Č .; Ř íhová, 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 (5), 3050−3061. (22) Ulbrich, K.; Etrych, T.; Chytil, P.; Jelínková, M.; Ř íhová, B. Antibody-targeted polymer-doxorubicin conjugates with pH-controlled activation. J. Drug Targeting 2004, 12 (8), 477−489. (23) Etrych, T.; Jelínková, M.; Ř íhová, B.; Ulbrich, K. New HPMA copolymers containing doxorubicin bound via pH-sensitive linkage: synthesis and preliminary in vitro and in vivo biological properties. J. Controlled Release 2001, 73 (1), 89−102. (24) Šubr, V.; Koňaḱ , Č .; Laga, R.; Ulbrich, K. Coating of DNA/ poly(L-lysine) complexes by covalent attachment of poly[N-(2hydroxypropyl)methacrylamide]. Biomacromolecules 2006, 7 (1), 122−130. (25) Etrych, T.; Strohalm, J.; Chytil, P.; Č ernoch, P.; Starovoytova, L.; Pechar, M.; Ulbrich, K. Biodegradable star HPMA polymer conjugates of doxorubicin for passive tumor targeting. Eur. J. Pharm. Sci. 2011, 42, 527−539.

Dox suggest that the conjugate may be an efficient anticancer nanomedicine.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +420-296 809 231. Fax: +420-296 809 410. E-mail: [email protected]. ORCID

Tomás ̌ Etrych: 0000-0001-5908-5182 Author Contributions ‡

P.C. and M.S. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Czech Science Foundation (Grants 17-08084S and 17-13283S) and Ministry of Education, Youth and Sports of the Czech Republic within the National Sustainability Program I (Project POLYMAT LO1507).

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ABBREVIATIONS USED Đ, dispersity; DMSO, dimethyl sulfoxide; Dox, doxorubicin; GPC, gel permeation chromatography; HPLC, high-performance liquid chromatography; HPMA, N-(2-hydroxypropyl) methacrylamide; LTS, long-term survivor; Mw, weight-average molecular weight; NMR, nuclear magnetic resonance spectroscopy; PAMAM, poly(amido amine); Opb-Chol, cholest-5-en3β-yl 4-(2-oxopropyl)benzoate; TFA, trifluoroacetic acid; TT, thiazolidine-2-thione

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REFERENCES

(1) Kopeček, J.; Kopečková, P. HPMA copolymers: Origins, early developments, present, and future. Adv. Drug Delivery Rev. 2010, 62 (2), 122−149. (2) Ulbrich, K.; Holá, K.; Šubr, V.; Bakandritsos, A.; Tuček, J.; Zbořil, R. Targeted Drug Delivery with Polymers and Magnetic Nanoparticles: Covalent and Noncovalent Approaches, Release Control, and Clinical Studies. Chem. Rev. 2016, 116 (9), 5338−5431. (3) Quan, L. D.; Zhang, Y. J.; Crielaard, B. J.; Dusad, A.; Lele, S. M.; Rijcken, C. J. F.; Metselaar, J. M.; Kostková, H.; Etrych, T.; Ulbrich, K.; Kiessling, F.; Mikuls, T. R.; Hennink, W. E.; Storm, G.; Lammers, T.; Wang, D. Nanomedicines for Inflammatory Arthritis: Head-to-Head Comparison of Glucocorticoid-Containing Polymers, Micelles, and Liposomes. ACS Nano 2014, 8 (1), 458−466. (4) Maeda, H.; Wu, J.; Sawa, T.; Matsumura, Y.; Hori, K. Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review. J. Controlled Release 2000, 65 (1−2), 271−284. (5) Matsumura, Y.; Maeda, H. A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs. Cancer Res. 1986, 46 (12 Part 1), 6387−92. (6) Ř íhová, B.; Strohalm, J.; Prausová, J.; Kubácǩ ová, K.; Jelínková, M.; Rozprimová, L.; Šírová, M.; Plocová, D.; Etrych, T.; Šubr, V.; Mrkvan, T.; Kovár,̌ M.; Ulbrich, K. Cytostatic and immunomobilizing activities of polymer-bound drugs: experimental and first clinical data. J. Controlled Release 2003, 91 (1−2), 1−16. (7) Fang, J.; Nakamura, H.; Maeda, H. The EPR effect: Unique features of tumor blood vessels for drug delivery, factors involved, and limitations and augmentation of the effect. Adv. Drug Delivery Rev. 2011, 63 (3), 136−151. (8) Fox, M. E.; Szoka, F. C.; Frechet, J. M. Soluble polymer carriers for the treatment of cancer: the importance of molecular architecture. Acc. Chem. Res. 2009, 42 (8), 1141−51. I


Article

Molecular Pharmaceutics (26) Etrych, T.; Chytil, P.; Jelínková, M.; Ř íhová, B.; Ulbrich, K. Synthesis of HPMA copolymers containing doxorubicin bound via a hydrazone linkage. Effect of spacer on drug release and in vitro cytotoxicity. Macromol. Biosci. 2002, 2 (1), 43−52. (27) Hirst, D. G.; Brown, J. M.; Hazlehurst, J. L. Enhancement of Ccnu Cyto-Toxicity by Misonidazole - Possible Therapeutic Gain. Br. J. Cancer 1982, 46 (1), 109−116. (28) Chytil, P.; Koziolová, E.; Etrych, T.; Ulbrich, K. HPMA Copolymer−Drug Conjugates with Controlled Tumor-Specific Drug Release. Macromol. Biosci. 2018, 18 (1), 1700209. (29) Nakamura, H.; Etrych, T.; Chytil, P.; Ohkubo, M.; Fang, J.; Ulbrich, K.; Maeda, H. Two step mechanisms of tumor selective delivery of N-(2-hydroxypropyl)methacrylamide copolymer conjugated with pirarubicin via an acid-cleavable linkage. J. Controlled Release 2014, 174 (0), 81−87. (30) Etrych, T.; Strohalm, J.; Šírová, M.; Tomalová, B.; Rossmann, P.; Ř íhová, B.; Ulbrich, K.; Kovár,̌ M. High-molecular weight star conjugates containing docetaxel with high anti-tumor activity and low systemic toxicity in vivo. Polym. Chem. 2015, 6 (1), 160−170. (31) Etrych, T.; Šírová, M.; Starovoytova, L.; Ř íhová, B.; Ulbrich, K. HPMA Copolymer Conjugates of Paclitaxel and Docetaxel with pHControlled Drug Release. Mol. Pharmaceutics 2010, 7 (4), 1015−1026. (32) Mrkvan, T.; Šírová, M.; Etrych, T.; Chytil, P.; Strohalm, J.; Plocová, D.; Ulbrich, K.; Ř íhová, B. Chemotherapy based on HPMA copolymer conjugates with pH-controlled release of doxorubicin triggers anti-tumor immunity. J. Controlled Release 2005, 110 (1), 119−29. (33) Šírová, M.; Mrkvan, T.; Etrych, T.; Chytil, P.; Rossmann, P.; Ibrahimová, M.; Kovár,̌ L.; Ulbrich, K.; Ř íhová, B. Preclinical Evaluation of Linear HPMA-Doxorubicin Conjugates with pHSensitive Drug Release: Efficacy, Safety, and Immunomodulating Activity in Murine Model. Pharm. Res. 2010, 27 (1), 200−208. (34) Šírová, M.; Kabešová, M.; Kovár,̌ L.; Etrych, T.; Strohalm, J.; Ulbrich, K.; Ř íhová, B. HPMA Copolymer-Bound Doxorubicin Induces Immunogenic Tumor Cell Death. Curr. Med. Chem. 2013, 20 (38), 4815−4826.

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Bloodstream Stability Predetermines the Antitumor Efficacy of Micellar

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