Blood stream stability predetermines the anti-tumor efficacy of micellar

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Blood stream stability predetermines the anti-tumor efficacy of micellar polymer-doxorubicin drug conjugates with pH-triggered drug release Petr Chytil, Milada Sirova, Júlia Kudlá#ová, Blanka Rihova, Karel Ulbrich, and Tomáš Etrych Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00156 • Publication Date (Web): 15 Mar 2018 Downloaded from http://pubs.acs.org on March 16, 2018

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Molecular Pharmaceutics

Blood stream stability predetermines the anti-tumor efficacy of micellar polymer-doxorubicin drug conjugates with pH-triggered drug release Petr Chytil1‡, Milada Šírová2‡, Júlia Kudláčová1, Blanka Říhová2, Karel Ulbrich1, Tomáš Etrych1* 1

Institute of Macromolecular Chemistry, Czech Academy of Sciences, Heyrovského nám. 2, 162

06 Prague 6, Czech Republic 2

Institute of Microbiology, Czech Academy of Sciences, Vídeňská 1083, 142 20 Prague 4, Czech

Republic ‡

These authors contributed equally.

*Corresponding author: tel.: +420-296 809 231; fax: +420-296 809 410 E-mail address: [email protected] KEYWORDS Polymer micelles; degradation; stimuli response; HPMA copolymer, solid tumor treatment

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ABSTRACT

Herein, the biodegradable micelle-forming amphiphilic N-(2-hydroxypropyl) methacrylamide (HPMA)-based polymer conjugates with the anti-cancer drug doxorubicin (Dox) designed for enhanced tumor accumulation were investigated, and the influence of their stability in the blood stream 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 blood stream 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 conjugates biodistribution on the stability of the micellar structure was observed in neutral, blood streammimicking, 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, non-degradable, high-molecular-weight star-like HPMA copolymer-Dox conjugate, which was highly efficient in the treatment of solid tumors in mice. Based on the results, we conclude that the blood-stream 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 anti-tumor activity.


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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, non-toxicity, and nonimmunogenicity.1, 2 A typical application is the treatment of cancer and inflammatory diseases.2, 3 The nano-sized 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 anti-cancer drugs, mainly cytostatic agents (e.g., doxorubicin (Dox), pirarubicin, or docetaxel), to HPMA-based copolymers, the tumor accumulation of the drugs can be significantly enhanced. Following enzymolytic or pH-triggered 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 (approx. 50,000 g/mol 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 approx. 500,000 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 long-term accumulation in the body after fulfilling their role


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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 selfassembly 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 Afterwards, similar amphiphilic polymer-Dox conjugates containing pH-sensitive hydrazone bond in spacers between the hydrophilic polymer carrier and hydrophobic cholesterol moieties were synthesized and their physico-chemical 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 in 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 physicochemical-dependent transport of nanoparticles through the tumor


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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 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 anti-cancer activity. We focused our study on the biodistribution, tumor accumulation and anti-cancer activity of the biodegradable micelle-forming polymer-Dox conjugates with respect to the detailed polymer carrier structure.

Materials and Methods Chemicals


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We purchased 1-aminopropan-2-ol, methacryloyl chloride, 6-aminohexanoic acid, methyl 6aminohexanoate hydrochloride, hydrazine hydrate, tert-butyl carbazate, trifluoroacetic acid (TFA), N,N´-dicyclohexylcarbodiimide, 4-(dimethylamino)pyridine, cholesterol, cholest-4-en-3one, 5α-cholestanone, dicyclohexylcarbodiimide, dimethyl sulfoxide (DMSO), N,Ndiisopropylethylamine, 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-AhxNHNH2) 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 cholest5-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 reverse-phase Chromolith Performance RP-18e (4.6 × 100 mm) column with photo diode array detection (Shimadzu SPD-M20A). 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


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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-Ahx-NHNH-Boc)-TT with the terminal thiazolidine-2-thione (TT) chain end group onto the 2nd generation PAMAM dendrimer containing terminal amino groups followed by hydrazide group deprotection as described in Ref23. The semitelechelic copolymer was prepared by free radical polymerization 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 Methods


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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 was determined spectrophotometrically, and the absence of unbound drug in the polymer-drug conjugates was verified by HPLC. The molecular weights (Mw) and dispersity (Ð) of the polymer precursors and their drug conjugates were determined by HPLC equipped with a GPC column (TSKgel Super SW3000, 300 x 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 Ð 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. Table 1 Characteristics of the HPMA copolymer-Dox conjugates


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Conjugate No.

Structure

Hydrophobic moiety

Mw (g/mol)a

Ða

Drug content RH (nm) (wt.%)

P1-Dox

micellar

cholest-4-en-3-one

25,500

1.8

8.1

14.8

P2-Dox

micellar

Opb-Chol

38,000

1.8

9.4

12.5

P3-Dox

micellar

5α-cholestanone

24,500

1.9

8.2

13.0

SP-Dox

star-like

-

250,000

1.9

9.5

12.6

a)

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

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 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 (U.S.A), 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


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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 T-cell 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 (s.c.) transplanted at day zero with 1×105 EL4 cells and were treated with the polymer conjugates injected intravenously (i.v.) 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 eq. 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 s.c. at day 120 after the first tumor transplantation and were left untreated. The tumor growth and survival were monitored.


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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 tumor-bearing mice were administered i.v. with polymer-Dox conjugates, a single dose of 10 mg Dox eq./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 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 reversedphase 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


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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, and 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

Blood stream stability predetermines the anti-tumor efficacy of micellar

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