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Biodegradable micellar HPMA-based polymer-drug conjugates with betulinic acid for passive tumor targeting Ekaterina A. Lomkova, Petr Chytil, Olga Janoušková, Thomas Mueller, Henrike Lucas, Sergey K. Filippov, Olga Trhlíková, Pavel A. Aleshunin, Yury A. Skorik, Karel Ulbrich, and Tomas Etrych Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00947 • Publication Date (Web): 16 Sep 2016 Downloaded from http://pubs.acs.org on September 18, 2016

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Biodegradable micellar HPMA-based polymer-drug conjugates with betulinic acid for passive tumor targeting Ekaterina A. Lomkova1,2, Petr Chytil1*, Olga Janoušková1, Thomas Mueller3, Henrike Lucas4, Sergey K. Filippov1, Olga Trhlíková1, Pavel A. Aleshunin5, Yury A. Skorik2,6, Karel Ulbrich1, Tomáš Etrych1 1

Institute of Macromolecular Chemistry, The Czech Academy of Sciences, Heyrovsky Sq. 2, Prague 6, 162 06, Czech Republic

2

St. Petersburg State Chemical Pharmaceutical Academy, 14 Prof. Popov St., St. Petersburg 197022, Russian Federation

3

Martin-Luther-University Halle-Wittenberg, Department of Internal Medicine IV, Oncology and Haematology, Ernst-Grube-Str. 40, 06120 Halle (Saale), Germany 4

Martin-Luther-University Halle-Wittenberg, Institute of Pharmacy, AG Pharmaceutical Technology, Wolfgang-Langenbeck-Str. 4, 06120 Halle (Saale), Germany

5

St. Petersburg State Technological Institute (technical university), 26 Moskovsky Pr., St. Petersburg, 190013, Russian Federation

ACS Paragon Plus Environment

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Institute of Macromolecular Compounds of the Russian Academy of Sciences, 31 Bolshoy pr. VO, St. Petersburg 199004, Russian Federation

Keywords: N-(2-hydroxypropyl)methacrylamide (HPMA), polymeric micelles, drug delivery, betulinic acid, EPR effect.

Abstract

Here, we present the synthesis, physico-chemical and preliminary biological characterization of micellar polymer-betulinic acid (BA) conjugates based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer carriers, enabling the controlled release of cytotoxic BA derivatives in solid tumors or tumor cells. Various HPMA copolymer conjugates differing in the structure of the spacer between the drug and the carrier were synthesized, all designed for pH-triggered drug release in tumor tissue or tumor cells. The high molecular weight of the micellar conjugates should improve the uptake of the drug in solid tumors due to the Enhanced permeability and retention (EPR) effect. Nevertheless, only the conjugate containing BA with methylated carboxyl groups enabled pH-dependent controlled release in vitro. Moreover, drug release led to the disassembly of the micellar structure, which facilitated elimination of the water-soluble HPMA copolymer carrier from the body by renal filtration. The methylated BA derivative and its polymer conjugate exhibited high cytostatic activity against DLD-1, HT-29 and HeLa carcinoma cell lines and enhanced tumor accumulation in HT-29 xenograft in mice.


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Introduction Triterpenoids, one of the most important classes of natural compounds, occur in many plant species. Derivatives of this class of compounds have been studied in the past few years due to their broad range of biological activities.1-6 In 1976, Trumbull et al.7 described the cytotoxic activity of a Vauquelinia corymbosa extract on the P-388 lymphocytic leukemia cell line. Betulin and betulinic acid [(3β)-3-hydroxy-lup-20(29)-en-28-oic acid; BA] were identified as active components in this extract.8, 9 The cytostatic properties of BA were verified in the early 1990s.10 It is widely recognized that BA has a broad spectrum of biological activities, including antiretroviral,

anti-bacterial,

anti-inflammatory,

anticancer,

anti-oxidant,

antimalarial,

and

anthelmintic.11-15 Unfortunately, BA and its metabolic precursor betulin are almost insoluble in water, which limits their pharmaceutical applications. Therefore, various derivatives of BA with improved properties were prepared and studied.16-21 The development of new drug delivery formulations based on polymer carriers has become an important pharmaceutical approach in recent decades. Most of these carrier systems have been primarily intended for use as carriers of anticancer or anti-inflammatory agents.22-24 Conjugation of a drug with a hydrophilic or amphiphilic polymer carrier or its incorporation into nanoparticle carriers significantly influences drug pharmacokinetics, e.g., it prolongs its circulation time in the blood and improves its solubility and bioavailability, thereby decreasing severe side effects and increasing therapeutic efficacy. Nano-scaled polymer carriers are preferentially accumulated in solid tumors due to the Enhanced Permeability and Retention (EPR) effect induced by the structural differences between healthy and malignant tissues.25 However, only a few articles have been published describing BA-polymer conjugates; e.g. the eight-arm-poly(ethylene glycol)-BA conjugate,26 or liposomal formulations 27, 28.


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The synthetic polymer carriers based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers have been intensively investigated as an efficient tool for controlled drug delivery to solid tumors.22, 29 Some conjugates of HPMA copolymers with drugs bound by biodegradable spacers, enabling controlled drug release in tumor cells, have already undergone clinical trials.30 An effective drug release mechanism is a prerequisite for an efficient tumor treatment. In recent years, polymer conjugates enabling the pH-triggered releases of anticancer agents (e.g., doxorubicin, taxanes or dexamethasone) from HPMA copolymer conjugates have been intensively studied.31-33 Here, the hydrazone bond is recognized as a pH-sensitive linkage because it is fairly stable at neutral pH, corresponding to blood pH, but hydrolyzes under mildly acidic conditions, corresponding to the environment in endosomes or lysosomes of tumor cells. The appropriate size and molecular weight of the polymer-drug conjugates are important for effective drug accumulation in tumors due to the EPR effect. Because polymers with molecular weights beyond the renal filtration limit (approx. 50000 g·mol-1 for water-soluble HPMA copolymers) cannot be eliminated from the body. Hence, polymer drug carriers exhibiting significant EPR effect should form supramolecular structures in order to increase passive targeting to solid tumors and, at the same time, allow the elimination of the carriers from the body after delivering their cargo.34 several HPMA-based polymer carriers that self-assemble into polymer micelles in aqueous solutions have been proposed and studied.35 For example, amphiphilic block copolymers with poly(lauryl methacrylate)36 or poly(ε-caprolactone)37 hydrophobic block were described. A recent study described properties of amphiphilic HPMA copolymers containing hydrophobic cholesterol molecules randomly distributed along polymer chains and their conjugates with doxorubicin bound by the hydrazone bond that self-assembled into micelles.38 They showed prolonged blood circulation, enhanced tumor uptake, and superior


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anticancer activity in the treatment of EL4 T-cell lymphoma.39 Similar HPMA copolymer bearing folate moieties randomly distributed along the polymer backbone were described.40 Recently, biodegradable micellar polymer-doxorubicin conjugates containing a hydrolysable spacer between the hydrophobic and hydrophilic parts were also described.41, 42 Biodegradability of the micellar carrier system should enable better control of the carrier elimination. This paper addresses the synthesis, physico-chemical characteristics, in vitro cytotoxicity, cellular uptake or apoptotic/ cell death changes and preliminary in vivo behavior of a new tumortargeted biodegradable micellar drug delivery system. This system is based on linear amphiphilic HPMA copolymer conjugates of BA derivatives bound to the copolymer chain by the pHsensitive hydrazone bond, enabling intratumoral or intracellular drug release. The self-assembly of the conjugates into micellar structures in aqueous solution due to the hydrophobic character of BA was an integral part of this study. The elimination of the carrier from an organism via control of the transformation of the amphiphilic micelle-forming polymer-drug conjugate into an excretable water-soluble HPMA copolymer based on pH-dependent hydrophobic drug release was regarded as one of the most important goals of this study. Experimental Section Chemicals 1-Aminopropan-2-ol,

2,2′-azoisobutyronitrile

(AIBN),

methacryloyl

chloride,

6-

aminohexanoic acid, 4-(dimethylamino)pyridine (DMAP), dicyclohexylcarbodiimide (DCC), N,N-diisopropylcarbodiimide (DIPC), 2,4,6-trinitrobenzene-1-sulfonic acid (TNBSA), levulinic acid (LEV), methyl iodide, N,N-diisopropylethylamine (DIPEA), cesium carbonate anhydrous, tert-butyl carbazate, trifluoroacetic acid and cyano-2-propyl benzodithioate were purchased from Sigma-Aldrich. Betulinic acid (BA) and betulonic acid (BoA) were purchased from Betulinines


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(Czech Republic). 3-Acetyl acrylic acid (AA) was purchased from Alfa Aesar. Alexa Fluor 488 NHS ester was purchased from Life Technologies. Dyomics782 NHS ester was purchased from Dyomics GmbH, Jena, Germany. All other chemicals and solvents were of analytical grade. The solvents were dried and purified by conventional procedures. Synthesis of the monomers and BA derivatives HPMA, m.p. 69 – 70°C, elemental analysis (calc./found): C 58.72/58.98, H 9.15/9.18, N 9.78/9.82, 6-methacrylamidohexanohydrazide (MA-Ahex-NHNH2), m.p. 79 – 81°C, elemental analysis (calc./found): C 56.32/56.49; H 8.98/8.63; N 19.70/19.83 and N-(tert-butoxycarbonyl)2-(6-methacrylamido hexanoyl)hydrazine (MA-Ahex-NHNH-Boc), m.p. 114 - 116°C; elemental analysis (calc./found): C 57.70/58.26; H 8.33/8.95; N 13.46/13.25 monomers were prepared as described previously.43-45 BA levulinate [(3β)-3-(4-oxopentanoyloxy)-lup-20(29)-en-28-oic acid; BA-LEV] was prepared by esterification of LEV with the hydroxyl group of BA using the carbodiimide coupling method. 339 mg of DCC (1.642 mmol), 191 mg of LEV (1.642 mmol), 286 µL of DIPEA (1.642 mmol) and a few crystals of DMAP were dissolved in 700 µL of tetrahydrofuran (THF) and cooled at -18°C for 20 min. 250 mg of BA (0.547 mmol) were dissolved in 1 ml of THF and cooled at -18°C for 20 min. Both solutions were mixed and the reaction mixture was cooled at -18°C for 1 h, then at 4°C for 20 h. The course of the reaction was monitored by reversed-phase HPLC as described below. Unreacted DCC was quenched by addition of 63 µL of acetic acid to the reaction mixture. The precipitated N,N′-dicyclohexylurea was filtered off, and the product was purified by column chromatography (silica gel 60) using an ethyl acetate/hexane mixture (1:1 v/v). The product purity was verified by HPLC and 1H-NMR. Yield: 92 %; (C35H54O5)n (554.80)n: Calcd. C 75.77, H 9.81, Found C 74.90, H 10.07; 1H NMR (600


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MHz, THF-d8) δ 10.70 (s, 1H, COOH), 4.70 (d, 1H, C=CH), 4.56 (d, 1H, C=CH), 4.46 (dd,1H, CH-O), 3.05 (td, 1H, CH), 2.68 (t, 2H, COCH2CH2CO2), 2.56 – 2.43 (m, 2H, COCH2CH2CO2), 2.37 (td, 1H, CH), 2.23 (dt 1H, CH), 2.07 (s, 3H, CH3CO), 1.90 (q, 2H, CH2), 1.68 (s, 3H, CH3), 1.65 – 1.04 (m, 20H, BA-cycle), 1.01 (s, 3H, CH3), 0.97 (s, 3H, CH3), 0.88 (s, 3H, CH3), 0.84 (d, J = 2.4 Hz, 6H, C(CH3)2); APCI−MS: m/z = 553.6 (M−H). BA 3-acetyl acrylate [(3β)-3-(4-oxo-2-pentenoyloxy)-lup-20(29)-en-28-oic acid; BA-AA] was prepared by esterification of AA with the hydroxyl group of BA using the carbodiimide coupling method. 207 mg of DIPC (1.642 mmol), 187 mg of AA (1.642 mmol), 286 µL of DIPEA (1.642 mmol) and a few crystals of DMAP were dissolved in 700 µL of THF and cooled at -18°C for 20 min. 250 mg of BA (0.547 mmol) were dissolved in 1 mL of THF and cooled at -18°C for 20 min. The two resulting solutions were mixed and the reaction mixture was cooled at -18°C for 1 h, then at 4°C for 20 h. The course of the reaction was monitored by HPLC. The reaction mixture was thickened to an oily consistency and dissolved in THF. The pure product was obtained by HPLC using a semipreparative Chromolith SemiPrep RP-18e, 100 × 10 mm column at a flow rate of 5 mL·min-1 and photodiode array (PDA) detector SPD-M20A (Shimadzu, Japan); water – acetonitrile, gradient 0 – 100 % acetonitrile. The fractions containing the pure product were collected and evaporated to dryness under reduced pressure. The product purity was verified by HPLC and 1H-NMR. Yield: 48 %; (C35H52O5)n (552.78)n: Calcd. C 76.05, H 9.48, Found C 75.48, H 10.52; 1H NMR (600 MHz, THF-d8) δ 10.71 (s, 1H, COOH), 6.90 (d, 1H, C=CHCO), 6.66 (d, 1H, C=CHCO), 4.70 (d, 1H, C=CH), 4.60 (dd,1H, CH-O), 4.56 (d, 1H, C=CH), 3.06 (td, 1H, CH), 2.37 (td, 1H, CH), 2.29 (s, 3H, CH3CO), 2.23 (dt 1H, CH), 1.90 (q, 2H, CH2), 1.69 (s, 3H, CH3), 1.68 – 1.04 (m, 20H, BA-cycle), 1.02 (s, 3H, CH3), 0.98 (s, 3H, CH3), 0.91-0.90 (d, J = 2.4 Hz, 6H, C(CH3)2), 0.87 (s, 3H, CH3); APCI−MS: m/z = 551.4 (M−H).


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The methylated BA levulinate [methyl (3β)-3-(4-oxo-2-pentenoyloxy)-lup-20(29)-en-28-oate; BA-M-LEV] was prepared by methylation of BA-LEV. 200 mg of BA-LEV (0.360 mmol) were dissolved in 3 mL of DMSO, 141 mg of cesium carbonate anhydrous (0.433 mmol) were added to the solution of BA-LEV under stirring. After 10 min, 45 µL of methyl iodide (0.721 mmol) were added to the reaction mixture, which was then stirred for further 2 h. The course of the reaction was monitored by HPLC. Then the product was precipitated into distilled water, filtered and dried under vacuum to a constant weight. The product purity was verified by HPLC and GCFID. Yield: 82 %; (C36H56O5)n (568.83)n: Calcd. C 76.01, H 9.92, Found C 75.93, H 9.98; 1H NMR (600 MHz, THF-d8) δ 4.71 (d, 1H, C=CH), 4.57 (d, 1H, C=CH), 4.45 (dd,1H, CH-O), 3.61 (s, 3H, COOCH3), 3.02 (td, 1H, CH), 2.68 (t, 2H, COCH2CH2CO2), 2.47 – 2.43 (m, 2H, COCH2CH2CO2), 2.31 (td, 1H, CH), 2.21 (dt 1H, CH), 2.07 (s, 3H, CH3CO), 1.85 (q, 2H, CH2), 1.69 (s, 3H, CH3), 1.67 – 1.06 (m, 20H, BA-cycle), 1.00 (s, 3H, CH3), 0.94 (s, 3H, CH3), 0.88 (s, 3H, CH3), 0.85-0.84 (d, J = 2.4 Hz, 6H, C(CH3)2); MALDI-TOF MS (591.4, M+Na). Synthesis of the polymer precursors and drug conjugates A random copolymer of HPMA with MA-Ahex-NHNH2 (polymer 1) was prepared by radical copolymerization in methanol as described by Etrych et al.43 The polymer-drug conjugates with BA derivatives differing in their BA content were prepared by the reaction of free hydrazide groups of polymer 1 with keto group-containing derivatives of BA; i.e., BoA (2a – e), BA-LEV (3a – d), BA-AA (4a) and BA-M-LEV (5a – c) (see Scheme 1). A representative synthesis of polymer conjugate 2a is as follows: a 15 wt% methanolic solution of polymer 1 (100 mg; 54.1 µmol of hydrazide groups) with 40 µL of acetic acid was added to the calculated amount of BA derivative (3.1 mg; 10.2 µmol) and stirred for 18 h at room temperature. The polymer was isolated by precipitation into an excess of ethyl acetate and


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purified by precipitation from methanol in ethyl acetate. The final conjugate was filtered off and dried under vacuum until a constant weight was achieved.

HN

O

+

AIBN methanol, 60°C, 17 h

x

HN

HN

HO

O

O

HN

HN

HN

HN

O NH

NH2

NH2

x

HO

O NH

O

O

O

R=O methanol, acetic acid, r.t., 18 h

HO

O

/

/

/ O

O NH N

NH NH2

R polymer 1

conjugates 2a - 5c

R=O is H

H OH

H O

H

O

O

H

O

O

H for conjugates 4a - d

H O O

H O

O

H

for conjugates 2a - e

H

OH

H

O

H

O

O O

H

OH

H

H O

for conjugate 3a

H

O

for conjugates 5a - c

Scheme 1. Scheme of the synthesis of the conjugates with BA derivatives prepared using free radical polymerization.

The polymer conjugate labeled by fluorescent dye Alexa Fluor 488 (conjugate 5a-dye) was synthesized by the reaction of Alexa Fluor 488 NHS ester with the hydrazide groups of conjugate 5a. In total, 30 mg of conjugate 5a and 0.3 mg of Alexa Fluor 488 NHS ester were


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dissolved in 0.3 mL of methanol and stirred for 18 h at room temperature in the dark. The polymer was purified by gel filtration on a Sephadex LH-20 column (GE Healthcare Life Sciences) in methanol. The polymer-dye conjugate was isolated by precipitation into ethyl acetate, and the precipitate was filtered off and dried under vacuum until a constant weight was achieved. A a control polymer 1-dye labeled by fluorescent dye Alexa Fluor 488 was prepared from polymer 1 and purified as described above. 1.

S

O HN

CN

/

S

tert.-butyl alcohol/DMSO, 70°C, 16 h

x

HO

3. Trifluoroacetic acid (95%), r.t., 5 min

O

+

HN

HN

HN

2. AIBN, DMSO, 70°C, 4 h O

O

O

NH O

HN

HO

NH

polymer 6

O O

NH2

BA-M-LEV methanol, r.t. 18 h

cholest-4-en-3-one methanol, r.t. 18 h

/

/ HN

/

/ O

O

O

HN

HN

x

HN

HN

HO

O

O

O

HN

x

HO

O

O

O

O

NH

NH N

NH N

NH2

NH NH2

O O H

H H

H

H

H

H H O conjugate 7

O conjugate 8


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Scheme 2. Scheme of the synthesis of the conjugates with BA-M-LEV and cholest-4-en-3-one prepared using the RAFT polymerization. A low dispersed polymer precursor bearing hydrazide groups randomly distributed along the polymer

backbone

(polymer

6)

was

prepared

by

controlled

radical

Reversible

addition−fragmentation chain-transfer (RAFT) polymerization of HPMA and MA-Ahex-NHNHBoc using cyano-2-propyl benzodithioate as chain transfer agent, followed by the removal of dithiobenzoate groups and deprotection of hydrazide groups as described previously (see Scheme 2 for the synthesis).46 The conjugate 6-dye was prepared by labeling of the precursor with the near-infrared (NIR) fluorescent dye Dyomics782 by the reaction of Dyomics782 NHS ester with hydrazide groups of the polymer precursor according the procedure described for conjugate 5adye. Polymer 6 was also used for the synthesis of polymer conjugate bearing BA-M-LEV (conjugate 7) bound by hydrazone bond in the same drug content as its analogue with broad dispersity (conjugate 5a). For comparison of behavior of polymer micelles in vivo also polymer bearing similar content of cholest-4-en-3-one (conjugate 8) was prepared from polymer 6 according ref.42. Both conjugates 7 and 8 were labeled by NIR dye Dyomics782 (conjugate 7dye and 8-dye) and used for the biodistribution study. Purification and characterization of the derivatives and their polymer conjugates The purity of the monomers and BA derivatives was determined by 1H-NMR (DMSO-d6 or CDCl3; 300 MHz or 600 MHz using DPX 300 or AVANCE III 600 US+ instruments, respectively; Bruker, USA), HPLC using reversed-phase Chromolith High Resolution RP-18e, 4.6 × 100 mm or Chromolith High Resolution RP-8e, 4.6 × 100 mm column (Merck, Germany), and SPD-M20A photodiode array detector (PDA) (Shimadzu, Japan) with a linear gradient of water – acetonitrile (0 − 100% acetonitrile) in the presence of 0.1% trifluoroacetic acid for


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monomers with a flow rate of 5 mL·min-1, and GC-FID performed on a Perkin-Elmer AutoSystem gas chromatograph using a PE-Carbowax 20M column 25 m length, 0.25 mm i.d., 0.25 µm film thickness. The molecular masses of BA derivatives were determined using mass spectrometry performed on an LCQ Fleet mass analyzer with atmospheric pressure chemical ionization (APCI MS) (Thermo Fisher Scientific, USA) or by using Matrix Assisted Laser Desorption Ionization – Time of Flight (MALDI-TOF) performed on a Bruker BIFLEX III (Bruker Daltonics, USA). The molecular weights of the polymers and their conjugates were determined using a Shimadzu HPLC system equipped with a gel permeation chromatography (GPC) column (TSKgel G3000SWxl, 300 × 7.8 mm; 5 µm), PDA, refractive index (RI) Optilab®-rEX and multiangle light scattering (MALS) (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 0.5 mL·min-1). The content of hydrazide-terminated side chains in the polymer precursor was determined by a modified TNBSA assay, as previously described.47 The molar absorption coefficient ε500 = 17200 L·mol-1·cm-1 (λ = 500 nm) estimated for the model reaction of MA-Ahex-NHNH2 with TNBSA was used. The content of BA-LEV, BoA, and BA-M-LEV in the conjugates was determined by HPLC analysis after complete hydrolysis of the polymer conjugates in HCl solution (pH = 2.0) for 3 h at 37°C. The solutions containing the hydrolyzed BA-LEV conjugates were freeze-dried, and the resulting solid was suspended in THF. The amount of BA-LEV was determined by reversedphase HPLC as described above. The solutions containing hydrolyzed BoA and BA-M-LEV conjugates were extracted with 1 mL of chloroform, and the organic phase was removed and


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dried. The released BoA and BA-M-LEV were dissolved in THF, and the amounts were determined by reversed-phase HPLC. The content of BA-AA in the conjugate was determined by 1

H-NMR spectrometry. As the first step, the hydrodynamic radius (Rh) of the polymer conjugates was measured by

dynamic light scattering (DLS) in a phosphate buffer (5 mg·mL-1; pH 7.4, 0.1 M with 0.05 M NaCl) using a Nano-ZS instrument (ZEN3600, Malvern, UK). The intensity of scattered light was detected at angle θ = 173° using a laser with a wavelength of 632.8 nm. DLS data were evaluated using the DTS (Nano) program. The values were the mean of at least five independent measurements. The size distribution of conjugates during the polymer carrier disintegration study was measured by DLS in phosphate buffers at pH 5.0 or 7.4 (5 mg·mL-1; 0.1 M, with 0.05 M NaCl) at 37°C. To obtain detailed knowledge of the molecular weight (Mw) and the particle distribution in solution, measurements were carried out on an ALV instrument equipped with a 22 mW He-Ne laser at a broad angle range of 30°-150°. All measurements were performed in the same buffers as listed above. The resultant correlation functions were analyzed by REPES48, analytical software that provides the distribution function of hydrodynamic radii (G(Rh)). To account for the logarithmic scale on the Rh axis, all DLS distribution diagrams are shown in the equal area representation RhG(Rh).49 For all experiments, approximately 2 mL of the sample solution was filtered using a 0.22 µm PVDF filter and transferred to a sealed, dust-free light scattering cell. The temperature was controlled within 0.05°C. The apparent Rh of the nanoparticles was calculated using the Stokes-Einstein equation. The static light scattering (SLS) data were analyzed using the Zimm plot procedure. Toluene was used for calibration, and refractive index increment values were determined independently.


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The critical micellar concentration (CMC) values were determined by isothermal titration calorimetry (ITC) using a MicroCal iTC200 isothermal titration microcalorimeter. The ITC experiments were performed using 20 injections of polymer solution in the phosphate buffer at pH 7.4 above Ttr into the buffer (the titration volume was varied during the experiments between 0.3, 0.5, 1.0 and 2 µL; 120 s intervals). The thermograms were recorded and analyzed using Origin 7 software. In vitro release experiments The rates of BA derivative release from polymer conjugates were investigated in phosphate buffers at pH 5.0 or 7.4 (0.1 M, with 0.05 M NaCl) at 37°C using a polymer concentration of 5 mg·mL-1. The amount of released drug was determined by reversed-phase HPLC as described above, after either freeze-drying in the case of BA-LEV or extraction into chloroform in the case of BoA, BA-M-LEV, and BA-AA. The values were the mean of three independent experiments. The rate of water-soluble polymer carrier release from the micelles of polymer conjugate 5adye was investigated in phosphate buffer at pH 5.0 at 37°C using a polymer concentration of 5 mg·mL-1. At predetermined intervals (1, 2, 4 and 8 h), 0.1 mL of the solution was removed and diluted by 0.4 mL of water and ultrafiltrated using an Amicon Ultra-0.5 Centrifugal Filter Unit with Ultracel-50 membrane (cut off 50 kDa). The fluorescence of the polymer in the eluate was measured using a Jasco FP-6200 spectrofluorometer using excitation at 494 nm and emission at 517 nm. Immediately afterward, the size of the coil in the eluted polymer solution was determined by DLS using the Nano-ZS instrument. Cell lines The DLD-1 and HT-29 human colorectal carcinoma cell lines (ATCC, LGC Standards Sp. z.o.o. Poland) and HeLa human cervix carcinoma cell line (kindly provided by Dr. Melkova,


14

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First Faculty of Medicine, Charles University in Prague) were cultivated in RPMI, McCoy’s and DMEM, respectively (Thermo Scientific, CR). The media were supplemented with heat inactivated 10% fetal bovine serum (FBS), 100 U·mL-1 penicillin and 100 µg·mL-1 streptomycin. Cytostatic activity of BA derivatives and their polymer conjugates In total, 3·103 cells were seeded in 100 µL of media into 96-well flat-bottom plates (TPP, Czech Republic) 24 h before the addition of polymer-drug conjugates or the drug itself. The concentrations of BA and BA-derivatives varied in the range 0.1 – 100 µM or 0.25 – 250 µM of BA equivalent for drug conjugates. The cells were cultivated for 72 h in 5 % CO2 at 37°C. Then, 10 µL of alamarBlue® cell viability reagent (Life Technologies, Czech Republic) was added to each well and incubated for 4 h at 37°C. The active component of the alamarBlue reagent, resazurin, was reduced to the highly fluorescent compound resorufin only in viable cells. Its fluorescence was detected in a Synergy Neo plate reader (Bio-Tek, Czech Republic) using excitation at 570 nm and emission at 600 nm. As a control, cells cultivated in the medium without drugs or the polymer-drug conjugate were examined. Three wells were used for each concentration. The assay was repeated four times independently. The statistical analysis was performed using Origin 9 software (one-way ANOVA analysis and the Tukey test). Intracellular localization of polymer conjugates using Laser Scanning Confocal Microscopy (LSCM) DLD-1 or HeLa cell line (1·105 cells) were seeded in 1 mL of media on a 35 mm glass bottom dish with four chambers, a 20 mm micro well, and a# 1 cover glass (0.13−0.16 mm) (Bio-Port Europe, Czech Republic) one day before incubation with polymer conjugates. The polymers were added in final concentration of the dye (Alexa Fluor 488) 2 µg·mL-1 and incubated with cells for 24 h. Prior to the end of the incubation time (10 min) Hoechst 33342 (5 µg·mL-1,


15

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Thermo Scientific, Czech Republic) was added for the labeling of the nuclei. The Alexa Fluor 488 was excited at 488 nm and the emitted light was detected through the 500-600 nm filter. Hoechst 33342, dye labeling the nuclei, was excited at 405 nm and emitted light was detected through the 425 - 500 nm filter. The laser scanning confocal microscope Olympus IX83 with FV10-ASW software (Olympus, Czech Republic) was used to observe the fluorescence light. The samples were scanned with a 60x oil immersion objective Plan ApoN (1.42 numerical aperture). Determining induction of caspases activity and cell death changes by flow cytometry Conjugates 5a and 7, BA, BA-M-LEV and polymer 1 were assessed for they ability to induce caspases 3 and 7, which are activated during apoptotic changes in the cell using CellEvent™Caspase-3/7 Green Detection Reagent (Thermo Scientific, Prague, Czech Republic). Moreover, the cell death were assessed by labeling cells with Sytox®Blue Nucleic Acid Dye (Thermo Scientific, Prague, Czech Republic), which labeled death or dying cells. HeLa cells were seeded at 1·105 cells per 1 mL of RPMI medium for 24 h in 24 well plates. Conjugates, polymer, BA or BA-M-LEV were added at final BA or BA-M-LEV equivalent concentration of 100 µM and incubated with cells 24 h. Afterwards 5 µM CellEvent™Caspase-3/7 Green Detection reagent were added to the media and incubated with cells for 30 min in 5 % CO2 at 37°C. The cells were then detached from the well by incubation in detaching solution (0.5 % BSA, 10 mM EDTA, 100 mM NaCl, 20 mM HEPES, pH 7.4), centrifuged and labeled with 5 µM Sytox®Blue for 10 min. Samples were analyzed by FACS Verse (Becton Dickinson, Czech Republic) and FlowJo software (Tree Star, Ashland, OR, USA). The experiment was repeated three times. Mouse model


16

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All animal experiments complied with regional standards and regulations and were approved by the local authority in Saxony-Anhalt. The two human colorectal cancer cell lines HT-29 and DLD-1 were used in a subcutaneus (s. c.) growing tumor xenograft model in athymic nude mice for the in vivo evaluation of polymer conjugates 6-dye - 8-dye. All polymers were fluorescent duet to a labeling with Dyomics782. The athymic nude mice were purchased from the core facility Animal Husbandry of the Zentrum für Medizinische Grundlagenforschung (ZMG) of the medical faculty of the Martin Luther University Halle-Wittenberg, Halle, Germany. Mice were kept under controlled standard conditions (12 h day/night cycle, 24°C) with food and water ad libitum. Tumor cells were grown in RPMI-1640 medium supplemented with 10 % fetal calf serum and penicillin/streptomycin at 37°C and with 5 % CO2 enriched, water saturated atmosphere. For cell injection, the adherent growing cells were trypsinized, counted and aliquoted in phosphate buffered saline (PBS) to the desired cell density of 3.3·107 cells/mL. Nine adult male mice received a s. c. injection of 5·106 tumor cells in 150 µL PBS to establish the solid tumors. DLD1 cells were injected into the right and HT29 cells into the left flank of each mouse using a syringe with a fixed 27 G needle and without dead volume. Afterwards, the tumor dimensions as well as the body weight and mouse condition were monitored regularly. On day 13 after cell injection, 17 of 18 tumors had reached the desired volume of ≥ 200 mm³. Prior the injection of the different polymer formulations, mice were grouped with the aim of comparable tumor volumes. Groups size was n = 3. Polymer formulations were weighed to result polymer concentration of 15 mg·mL-1 in PBS. Then, the samples were sterile filtered through a syringe filter (0.22 µm pore size). Afterwards, each mouse was placed in a restrainer and received an intravenous (i. v.) injection via tail vein with a 30 G needle. The applied volume was 100 µL/mouse which corresponded to a dose of 1.5


17

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mg polymer per mouse independent from the actual mouse body weight. Mouse condition, body weight and tumor volume were monitored like before. On day 2 (48 h after polymer application), one individual from each groups was killed for ex vivo analysis of the biodistribution of the formulations. Therefore, organs were explanted and placed in a 12 well microtiter plate. Fluorescence imaging: Data acquisition and analysis Imaging was performed using a CRi Maestro Imaging device equipped with the Maestro software version 2.10.0. The mouse was anesthetized with inhalation narcosis using Isoflurane in Oxygen. Flow rate was set to 2 to 3 L·min-1 with an initial dosage of 4 to 5 % Isoflurane. For maintenance, the Isoflurane content was reduced to 1.5 to 3 % depending on the individual mouse. During the imaging procedure, the mouse was placed on a heating plate tempered at 35°C to prevent hypothermia. Initially, a grayscale picture was taken using IR illumination and 10 ms fixed exposure time. Then, the NIR filter settings were used for fluorescence imaging using automatic exposure time. The excitation range was 710 to 760 nm using the adequate band pass filter on top of the xenon light source. The emission was detected using the 800 nm long pass filter combined with the system inherent liquid crystal filter allowing the scanning of the spectral range from 780 to 950 nm in 10 nm steps. The detected fluorescence signals resulted in data sets called cubes with picture dimensions of 696 x 520 pixel and a 12-bit grayscale intensity value. The cubes were then unmixed into the different fluorophore signals (Dyomics782: polymer labeling signal, BG: black back ground signal, Mouse: sum signal of an untreated mouse or Plate: microtiter plate signal) using an a priori defined spectral library. The isolated Dyomics782 signals of the in vivo images were then analyzed for fluorescence intensity and the tumor accumulation value (TAV) calculated according ref.

50

. The isolated Dyomics782 signals

of the ex vivo organ plates was not quantified in numbers. Both types (in and ex vivo) of pure


18

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Dyomics782 signals were further processed using the Compare Image tool of the Maestro software (clip & stretch modus, jet black pseudo color scale). By that, intensity weighted pseudo color images were generated as well as the corresponding color scales. Using the GNU Image Manipulation Program (GIMP, version 2.9.2), overlays of the grayscale picture and the compare image were made. First, the grayscale picture was loaded. Then, the corresponding compare image was added as a new layer. For that layer, an alpha transparency channel was generated. Next, the color Black was set transparent. Finally, the new overlay was exported and saved in JPEG format. Results and discussion The synthesis, physico-chemical and preliminary in vitro biological properties of the new micellar polymer nanotherapeutics bearing triterpenoid drugs based on BA derivatives are described. Polymer-drug conjugates based on micellar HPMA copolymer carriers have a significant potential for the treatment of tumor diseases, especially because of their low blood clearance and enhanced accumulation in vascularized solid tumors (the EPR effect).35,

39

We

focused our study on the improvement of the polymer nanotherapeutics’ properties, namely drug content, size of the micellar drug delivery system in solution, and potential for safe polymer carrier elimination due to the disintegration of the micelles following pH-controlled drug release. Synthesis of polymer conjugates with BA derivatives Polymer 1 was synthesized by copolymerization of HPMA with a monomer containing hydrazide groups, according to the previously described procedure.43 The polymer carrier contained 5.3 mol% of hydrazide groups, which was sufficient for drug attachment. The groups were randomly distributed along the polymer chain; copolymerization parameters determined by Fineman – Ross method were ra=1.26 and rb=0.91 or by using Kelen – Tüdös method ra=1.26


19

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and rb=0.93. The final conjugates were obtained by the reaction of the polymer 1 hydrazide groups with one of the following keto groups: BoA (2a – e), BA-LEV (3a – d), BA-AA (4) or BA-M-LEV (5a – c) (See Scheme 1). The BA derivative content in the reaction mixture was varied (from 1.0 to 5.0 mol% for BoA, from 1.0 to 4.0 mol% for BA-LEV and from 1.0 to 3.0 mol% for BA-M-LEV; the units of mol% are related to the sum of monomer units in the copolymers) to study the influence of the BA derivative content on the behavior of the conjugates in aqueous solution (see Table 1). The yield of all drug-binding reactions was approximately 86 ± 4 %, which was independent of the type of derivative or its content. The attachment of BA derivatives to polymer 1 slightly increased its molecular weight, while its dispersity remained essentially unchanged (measured by GPC using RI and MALS detection of polymers dissolved in an organic solvent mixture). Our aim was to prepare polymer-drug conjugates with the hydrophobic drug loading still enabling the use of a simple procedure for micelle preparation, i.e., by dissolution in buffered saline. In the case of the polymer conjugate with BA-AA, the drug content was not optimized from this viewpoint because the drug release rate was insufficient for drug delivery purposes, as described below. Table 1. Physico-chemical characteristics of HPMA polymer conjugates with BA-derivatives. Polyme-

Derivative

rization techni-

TheoreNo.

que

Free radical

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Structure

Determi-

tical

ned

content

content

[mol%]

[mol%]

Mw c) [g·mol-1]

Ð

c)

Rh d) [nm]

1

–

0

0

27 500

1.78

4.7

1-dye a)

-

0

0

26 540

1.70

n.d.

2a

BoA

1.0

0.9

36 000

1.86

6.6

2b

BoA

2.0

1.8

39 400

2.02

7.7


20

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Controlled RAFT

Page 21 of 47

2c

BoA

3.0

2.4

39 600

1.93

8.1

2d

BoA

4.0

3.2

43 000

1.92

13.8

2e

BoA

5.0

4.5

46 200

1.90

n.d.

3a

BA-LEV

1.0

0.6

31 100

1.98

8.6

3b

BA-LEV

2.0

1.5

37 000

2.00

9.6

3c

BA-LEV

3.0

2.4

33 200

1.77

12.3

3d

BA-LEV

4.0

3.6

34 500

1.77

n.d.

4

BA-AA

1.0

0.9

35 100

2.06

9.4

5a

BA-M-LEV

1.0

0.9

31 700

1.72

11.8

5a-dye a)

BA-M-LEV

1.0

0.9

30 530

1.84

10.1

5b

BA-M-LEV

2.0

1.7

38 500

1.96

42.4

5c

BA-M-LEV

3.0

2.7

36 300

1.93

n.d.

6

–

0

0

34 300

1.15

4.2

6-dye b)

–

0

0

n.d.

n.d.

n.d.

7

BA-M-LEV

1.0

0.9

38 700

1.19

14.6

7-dye b)

BA-M-LEV

1.0

0.9

n.d.

n.d.

n.d.

1.2

1.0

38 500

1.18

13.6

1.2

1.0

n.d.

n.d.

n.d.

8 8-dye b)

Cholest-4en-3-one Cholest-4en-3-one

a)

Labeled by fluorescent dye Alexa Fluor 488;

b)

Labeled by fluorescent NIR dye Dyomics782;

c)

Molecular weight (Mw) and dispersity (Ð) of polymers were determined using GPC with MALS and RI detection in a methanol : acetate buffer mixture (80:20 vol%). Values were not determined for samples with Dyomics782, but GPC traces correspond with that of polymer without the dye; d)

Hydrodynamic radius (Rh) was determined by DLS using Nano-ZS instrument. Rh values were not determined for samples with Dyomics782, Rh of conjugates 2e, 3d and 5c was not determined due to the insolubility of the conjugates in the phosphate buffer. Polymer precursor with hydrazide groups (polymer 6) was prepared by controlled radical RAFT polymerization, thus its dispersity was very low (Ð = 1.15), while its molecular weight


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remained similar to that of polymer 1. GPC chromatograms of both polymer precursors are shown in Figure S4 in Supporting Information. As expected the attachment of BA-M-LEV or cholest-4-en-3-one by hydrazone bond influenced neither molecular weight nor dispersity (. Physico-chemical properties of the polymer bearing cholest-4-en-3-one were studied in detail previously.41, 42 This polymer formed micelles in aqueous solutions by self-assembling and because of the stabilization of hydrazone bond the cholest-4-en-3-one was not released to high extent and stability of the micelles was pretty high, not ensuring the disassembly and removal of the carrier from the body after the delivery of the cargo.The labeling of polymers (1.2 wt% for polymer 6-dye, 1.4 wt% for conjugates 7-dye and 8-dye) did not affect dispersity of the conjugates. Self-assembly of polymer-drug conjugates in water The extent of polymer carrier accumulation in solid tumors due to the EPR effect depends on their size and molecular weight.51 We expected that the introduction of hydrophobic BA or its derivatives into the hydrophilic HPMA copolymer would lead to amphiphilic copolymers selfassembling into micelles in aqueous solution. The nanoparticle sizes determined by DLS expressed as hydrodynamic radius (Rh) are shown in Table 1. Even low BA derivative content (conjugates 2a, 3a, 4 and 5a) resulted in an increased Rh in comparison with that of the parent polymer (1). A further increase in the hydrophobic drug content resulted in an increase in micelle size. Nevertheless, polymer conjugates with the highest drug loading (2e, 3d and 5c) did not dissolve in the phosphate buffer, as the hydrophobic drug content exceeded the highest content still enabling an effective conjugate solubility. All other conjugates completely dissolved within 10 – 20 min under stirring and sonication. Similar behavior was reported previously for the conjugates of cholesterol and its derivatives with HPMA copolymers.39, 42


22

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The hydrophobicity of the drug derivative also influenced the behavior of the conjugates in a buffer. Although the conjugates bearing BoA or BA-LEV contained comparable amounts of the drug, sizes were slightly higher for the conjugates with BA-LEV because of the higher hydrophobicity of BA-LEV (compare conjugates 2c and 3c, or 2d and 3d). However, the methylation of the carboxylic group of BA-LEV resulted in an increase in the derivative’s hydrophobicity. Thus, the Rh of conjugate 5a (containing only 0.9 mol% of BA-M-LEV) was roughly the same as the Rh of conjugate 3c (containing 2.4 mol of BA-LEV, approximately 12 nm). Moreover, a further increase in the BA-M-LEV content resulted in the formation of larger aggregates (conjugate 5b) or prevented dissolution of the conjugate completely (conjugate 5c). The size of conjugates 7 (containing 0.9 mol% of BA-M-LEV, i.e. the same amount as conjugate 5a) and 8 (containing 1.0 mol% of cholest-4-en-3-one), showing both low dispersity, after self-assembly into the supramolecular structures (Rh = 14.6 or 13.6, respectively) were in accordance with values determined for their analogues with broader dispersity prepared by free radical polymerization (see Table 1 or ref.42). Interestingly, polydispersity index (PDI) of all of micellar systems, independently on method of synthesis and type of hydrophobic substituent, were about 0.15-0.25. The molecular weight (Mw) of the nanoparticles prepared from conjugates 2a, 3a and 5a was determined by SLS using a Zimm procedure (see Table 2). The presence of the second mode, which was attributed to formation of larger aggregates of released BA-derivative as described in the next subchapters, became more pronounced over time, hindering the precise Mw determination of conjugates 2a and 3a. Only in the case of conjugate 3a could Mw be calculated. The Mw of nanoparticles formed from conjugate 5a was comparable with that of cholesterolbearing HPMA-based polymer micelles described earlier.39, 42 The positive values of the second


23

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virial coefficient obtained from the SLS experiments on sample 1 imply that water is a good solvent for non-modified highly hydrophilic polymers. In contrast, the negative second virial coefficient A2 for samples 3a and 5a indicates a tendency toward aggregation and confirms that water is a thermodynamically bad solvent for BA-LEV-modified HPMA copolymer. Table 2. Properties of conjugates with BA-derivatives as determined by light scattering methods.

No.

Derivative

Mw [g·mol-1]

A2 Rh 3 -2 [mol·dm ·g ] [nm]

1

–

27 300

1.677·10-07

6

2a

BoA

-

-

7

3a

BA-LEV

56 200

-1.198·10-07

9

5a

BA-M-LEV

202 900

-3.324·10-08

11

Determination of the CMC for conjugate 5a by ITC was performed at 37°C (see Figure S1 in the supporting material). Analysis of the experimental data revealed a CMC of 0.12 – 0.15 mg·mL-1 for the conjugate at the inflexion midpoint. The obtained value is comparable with the CMC of the cholesterol-bearing micelles. The similar size, stability and solution properties of conjugate 5a with those of the previously described in vivo, highly effective, cholesterolcontaining conjugates allows expectations of the same tumor accumulation as found with those conjugates. Release of BA derivatives from the polymer-drug conjugates As mentioned earlier, the pH-dependent controlled release of BA derivatives from the conjugates was one of the aims of the present study. The pH-sensitive hydrazone bond between the drug derivative and the polymer chain was expected to provide controlled drug derivative release, resulting in targeted drug activation (in the tumor and tumor cells), more favorable drug


24

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derivative pharmacokinetics and its better bioavailability, as had been shown for the cholesterolbased conjugates with doxorubicin. Moreover, in the BA-derivative system, it was expected that the controlled release of BA derivatives from the supramolecular structure would also promote the disassembly of nanoparticles, leading to the elimination of the polymer carrier from the body following the site-specific drug delivery and drug release. Reaction with the free hydrazide groups of polymer precursor 1 and formation of the hydrazone bond requires the presence of a keto group in the drug structure. Unfortunately, BA does not have any such keto group, and thus, it needs to be derivatized before conjugation. Several BA derivatives containing keto groups were selected or designed for attachment to the polymer backbone. BoA (commercially available) and BA-LEV, selected on the basis of previous work,31, 32 were used as suitable candidates with expected pH-sensitive release (as had been shown for the release of taxanes or dexamethasone from their polymer conjugates with similar carrier and spacer structures).


25

Biomacromolecules

A

100

Released BoA (%)

80 60 40 20 0 0

2

4

6

8 10 12 14 16 18 20 22 24 Time (h)

B

Released BA-LEV (%)

100 80 60 40 20 0 0

2

4

6

8 10 12 14 16 18 20 22 24 Time (h)

C

100 Released BA-M-LEV (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 47

80 60 40 20 0 0

2

4

6

8 10 12 14 16 18 20 22 24 Time (h)

Figure 1. Release of BA derivatives from their respective polymer conjugates during incubation in phosphate buffers at 37°C: A) release of BoA from conjugate 2a (●── pH 5, ▲- - - pH 7.4), B) release of BA-LEV from conjugate 3a (●── pH 5, ▲- - - pH 7.4), C) release of BA-M-LEV from conjugates 5a (C; ●── pH 5, ▲- - - pH 7.4) and 5b (C; ●── pH 5, ▲- - - pH 7.4). No free drug release was observed.


26

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Biomacromolecules

The release rate of the BA derivatives from the conjugates was studied at 37°C in a phosphate buffer at pH 5.0, modeling the environment of the endosomes of target cells, and at pH 7.4, modeling the environment of blood vessels. No free acid (drug) was detected in an incubation media. A significant difference in the release rates was expected, with a much higher rate anticipated in the mildly acidic environment. Both BoA and BA-LEV were liberated quickly (60 – 70 % within 2 h incubation) from their respective polymer conjugates (2a and 3a) at pH 5.0 (see Figures 1A and 1B), in accordance with our previous findings. Unfortunately, the drug derivative release rates at pH 7.4 were similar to those at pH 5.0, indicating the absence of pHsensitive control in the drug release. The carboxylic group (C28) in the BoA and BA-LEV structures most likely protonated the hydrazone bond and thereby accelerated its hydrolysis rate, even at pH 7.4. Similar behavior was observed in the case of doxorubicin release from the HPMA copolymer conjugates bearing a small number of carboxylic groups in the polymer carrier chain.52 Two strategies were proposed to overcome the impact of the carboxylic groups on the hydrazone bond hydrolysis and to enable achievement of pH-controlled drug release. First, a 3(acetyl)acrylic acid spacer was used instead of levulinic acid. Previously, it has been shown that conjugates of HPMA copolymers modified with a paclitaxel 3-(acetyl)acrylate derivative show a very low release of the drug (less than 10% within 24 h) at both pH 7.4 and pH 5.0.31 The authors proposed that the presence of the double bond in the spacer between the drug and the polymer carrier was responsible for such slow release. The presence of the double bond in the βposition to the hydrazone bond and the shift in the electron density between the two nitrogens was expected to decrease the rate and influence pH-dependence in BA release if a 3-


27

Biomacromolecules

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Page 28 of 47

(acetyl)acrylate spacer is used. Unfortunately, in this case, almost no BA-AA (less than 1 %) was released from conjugate 4 within 24 h at both pH levels (data not shown). Another idea to influence the pH-dependence of the drug release consisted of the methylation of the carboxylic group in the BA-LEV structure. The drug release rate of BA-M-LEV from conjugates 5a and 5b at pH 5.0 resulted in almost the same release rate as was observed previously in the case of BA-LEV, i.e., approximately 60 % of the derivative was liberated within 2 h of incubation (Figure 1C). However, the drug release rate at pH 7.4 was much slower; less than 20 % of BA-M-LEV was released within 24 h of incubation. This result confirmed the hypothesis regarding the acceleration of the hydrazone bond hydrolysis in conditions mimicking blood circulation (pH 7.4), due to the presence of the carboxyl group in the BA structure. The protection of the carboxylic group by methylation therefore promoted the pH-sensitive controlled release of BA derivatives from the conjugate, which was a prerequisite for the sufficient therapeutic activity of polymer-drug conjugates. To conclude, the polymer conjugate containing a methylated BA derivative (BA-M-LEV) fulfilled the basic criteria for an efficient anticancer prodrug, i.e., stability during blood transportation and fast release of the active drug in the mildly acidic tumor tissue or inside the cells. In vitro disassembly of micellar polymer carriers The biodegradability of high-molecular-weight drug delivery systems is essential for practical application of these systems as effective carriers that are passively accumulating in solid tumors. Well-timed degradation and elimination of the carrier (its components or degradation products) from the organism prevents long-term accumulation of the carrier after the treatment and is safe for the organism. Comparably, the strategy of the controlled disassembly of the micellar polymer


28

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carriers resulting in short, soluble, non-degradable, synthetic polymer chains also fulfills the requirement for efficient carrier elimination. In our case, this strategy was based on the biodegradation of the spacers between polymer chains and hydrophobic drug derivatives to achieve effective micellar carrier disintegration and elimination subsequent to drug release. The direct measurement of particle size by DLS was performed during the incubation of conjugates 2d, 3c and 5a in phosphate buffers at pH 7.4 and 5.0 at 37°C. In the cases of BoA (conjugate 2d) and BA-LEV (conjugate 3c), the majority of nanoparticles belongs to polymer micelles as is illustrated by volume distribution at 0 h. Nevertheless, a fast aggregate formation was immediately observed at both pH levels from the outset of incubation (see Figures S2 and S3 in the supporting material). The fast releasing hydrophobic BA derivatives (Figures 1A and 1B) likely aggregated into large particles with an Rh exceeding 100 nm. Not only did these aggregates of the free drug form but also re-arranged polymer micelles with an Rh of approximately 30 – 50 nm were formed in all cases, especially during the first 8 h of incubation. In these micelles, the released BA derivative was incorporated into the hydrophobic core by non-covalent hydrophobic interaction thus increasing their size. Finally, nor polymer micelles, neither released polymer carrier were detected due to the high intensity of formed large particles. Similar results had been observed in the case of HPMA copolymers with cholesterol-keto derivatives bound by a hydrazone bond to the polymer carrier42 where the re-arrangement of the micelles was proved using time-dependent SAXS/SANS analysis.41 The same behavior as described above was also observed for conjugate 5a bearing BA-M-LEV when incubated at pH 5.0. In this case, the very fast formation of aggregates belonging to the released BA derivative was observed, as shown in Figure 2. The particle size grew quickly (see time-dependence of Rh) and the quantity of these particles continuously increased (see volume


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distribution) in accordance with the fast release of the BA-M-LEV at pH 5.0, as shown in Figure 1C. The behavior of conjugate 5a incubated in buffer at pH 7.4 was comparable with that of the earlier described cholesterol derivatives showing similar slower drug release rates at this pH.42 The size of the polymer micelles formed after the copolymer dissolution (initial Rh = 11 nm) was investigated by DLS within the first 4 h of incubation. After the first hour, nanoparticles with an Rh of approximately 30 nm belonging to the “re-arranged” polymer micelles were formed. Their size increased moderately to approximately 60 nm within 8 h and to almost 70 nm within 24 h of incubation. Unlike during incubation at pH 5.0, no aggregates with an Rh higher than 100 nm were detected at pH 7.4. Time-dependence of size 100

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Figure 2. Behavior of micellar conjugate 5a in buffers at pH 7.4 or 5.0 at 37 °C determined by DLS and expressed as the time dependence of Rh, evaluated from intensity distribution (left) or volume distribution (right) at selected time intervals ( means Rh values for polymer micelles,


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means Rh values for the “re-arranged” polymer micelles, mean Rh values for aggregates of BA derivative).

Unfortunately, the presence of released hydrophilic polymer carrier could not be evidenced by DLS because the high intensity of light scattered by the large particles (free drug aggregates) overlapped the signal of smaller particles. Thus, prior to the DLS evaluation, the released polymer carrier was separated from the sample of conjugate 5a and incubated for 22 h at pH 5.0 at 37°C using centrifugal filter units with a 50 kDa membrane cutoff. The size of the polymer fraction that penetrated the membrane was Rh = 5.7, indicating the presence of the only polymer carrier (unimer) released from the micellar conjugate. Afterward, a more detailed study was performed using the fluorescently labeled conjugate 5a-dye containing 0.3 wt% of Alexa Fluor 488 dye. Here, the size of the eluted polymer and the fluorescence intensity of the polymerbound dye after ultrafiltration were determined. The polymer carrier with an Rh of approximately 5 – 6 nm was released from the conjugate quickly within 8 h of incubation (see Figure 3). As expected, the release rate corresponded to the release rate of BA-M-LEV at pH 5.0, as described above. 100

10

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8

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Figure 3. Determination of fluorescently labeled polymer carrier released from conjugate 5a-dye after its incubation in buffer pH 5.0 at 37°C followed by ultrafiltration using a centrifugal filter unit with a 50 kDa membrane cutoff. Fluorescence intensity of the eluate was related to the total intensity of the sample (left axis) and the size of the eluent (right axis).

The results of the in vitro study showed that the micellar structure of the polymer conjugate containing BA-M-LEV (conjugate 5a) could disintegrate quickly under mildly acidic pH, modeling conditions inside endosomes/lysosomes of tumor cells, but it could disintegrate slowly at the neutral pH of blood. Thus, it can be expected that the polymer-drug conjugate will preferably accumulate in solid tumors, and after drug release inside the tumor, the polymer carrier should be eliminated from the body by renal filtration. In vitro cytotoxicity As shown above, all the studied conjugates released keto-derivatives of the drug, but not the drug itself, when incubated in aqueous solutions. The biological activities of BoA, BA-LEV, and BA-M-LEV were therefore compared with those of the parent BA. A cell viability assay was used as a classical approach for testing the cytotoxicity of BA and its derivatives to assess the direct effect of drugs on target cancer cells. HT-29, DLD-1 (both human colorectal adenocarcinoma) and HeLa cell lines are commonly used cell lines in the cytotoxicity tests of BA. The results, expressed as the half of maximum inhibitory concentration (IC50), are shown in Table 3. BA-LEV showed the highest cytotoxicity among all the tested compounds in all studied cell lines; IC50 values in DLD-1 and HT-29 cell lines were significantly (P < 0.05) lower than that of BA. BoA also showed slightly higher cytotoxic activity in comparison with BA, but the


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difference was significant in only some cell lines. As shown above, the methylation of the carboxylic group (C28) was necessary for the achievement of pH-dependent controlled drug release. It was reported that the methylation of BA and BoA resulted in the nearly complete loss of their biological activity; in particular, they were inactive against Semliki Forest virus replication.53 Nevertheless, BA-LEV, the most effective BA-derivative among those tested in the current study, was chosen for methylation to verify the published statement. While esterification (methyl ester) of BA-LEV decreased its cytotoxicity by around seven times in the DLD-1 cell line, its IC50 value decreased only two-fold in the HT-29 and HeLa cell lines. However, the methylated BA-M-LEV derivative was still sufficiently cytotoxic to all three cell types, with practically the same cytotoxicity as BA for the HT-29 and HeLa cell lines and only moderately lower cytotoxicity for the DLD-1 line. Attachment of BA-M-LEV to the polymer carrier (conjugate 5a) did not decrease its cytotoxicity, and the IC50 value for polymer-bound BA-MLEV in the HT-29 cell line was significantly lower than for BA itself, or for parent derivative BA-M-LEV. The results of this in vitro evaluation of the BA-derivative polymer conjugates provide a good basis for their further development for drug delivery applications. Table 3. The cytotoxicity of BA and its derivatives, IC50 ± SD (µM). IC50 ± SD of BA or BA-derivative [µM] Compound

Cell line DLD-1

HT-29

HeLa

BA

70.75 ± 0.01

87.05 ± 3.89

62.65 ± 12.17

BoA

33.47 ± 24.49

66.38 ± 24.11

59.28 ± 11.58

BA-LEV

15.42 ± 1.58*

33.13 ± 13.14*

37.90 ± 0.01

BA-M-LEV

105.19 ± 10.05*

68.70 ± 3.54

63.61 ± 1.55


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BA-M-LEV bound 64.20 ± 5.28 in conjugate 5a *)

27.72 ± 9.31*

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40.90 ± 11.98

Significant difference of BA-derivative in comparison with BA, P < 0.05.

Intracellular localization For the evaluation of intracellular localization of conjugate 5a labeled by fluorescent dye Alexa Fluor 488 (conjugate 5a-dye) in comparison with the labeled polymer 1-dye were used two cell lines DLD-1 and HeLa cells, mainly to demonstrated the difference between interaction of polymers with different cells (see Figure 4). The intracellular localization into the DLD-1 cells of both polymers did not show any significant differences. The signal of conjugate 5a-dye was weaker and the similar pattern was evaluated more clearly in HeLa cells. This can be ascribed to the higher size of the polymer micelles. The intracellular localization in HeLa cells of polymer 1dye were less spread around the nuclei than localization of conjugate 5a-dye which can correspond to utilization of different endocytic pathways.


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Figure 4. Intracelular localization of polymers labeled by Alexa Fluor 488 (green) in DLD-1 and HeLa cells after 24 h incubation evaluated by laser scanning confocal microscopy. Nuclei were labeled with Hoechst 33342 (blue).

Flow cytometry


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B)

caspase positive + death cells

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100 80 60 40 20 0

Axis Title

Figure 5. A) Example of analysis of caspase activation and cell death induction after treatment HeLa cells with free or polymer bound drug. Sytox®Blue stained death cells, CellEvent™ Caspase-3/7 Green Detection reagent detected activated Caspases 3/7. B) Comparison of death and caspase 3/7 activated cells versus live cells from FACS analysis (n=3).

Flow cytometry analysis was used to evaluate apoptotic and cell death changes which accompany the treatment with free drug and polymer conjugates in HeLa cells as an example illustrating the activity of prepared conjugates. Figure 5A shows cell death and caspase 3/7 activation after the treatment of HeLa cells for 24 h with final 100 µM of BA or BA-M-LEV equivalent. Lower final concentration of BA or BA-M-LEV (50, 25 µM) were also tested. The patterns were similar but less pronounced (data not shown). The dot plots are divided by four quadrants. Q1 corresponds to the dying and dead cells, Q2 corresponds to the double positive


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dead or dying cells and cells with activated caspases 3/7, Q3 corresponds to the cells with caspase activation only and Q4 corresponds to the live cells. As a control we used nontreated cells or cells incubated with polymer 1 without drug. These cells shows some percentage of dead and caspases positive cell, which can reflect the processes which have to be count during preparation of cells for experiment as detaching, centrifugation and resuspendation. These control samples, as also shown in Figure 5B, have significantly higher percentage of live cells than samples with conjugate or free drug treatment. The pattern of dying cells on dot plot looks very different with significantly lower amount of dying and caspases positive cells. Samples with BA shows specific pattern, where strict population of dead cells are expressed and also this sample has highest percentage of caspases positive cells. The dot plot of samples after the treatment with BA-M-LEV and both conjugates keep closed similarity of patterns with increasing percentage of dying/dead cells and lower percentage of caspases positivity than BA. These phenomena could be influence by changes in type of cell death which can be cause by conjugates of BA-M-LEV. Tumor accumulation study Preliminary tumor accumulation study of polymer-bound BA-M-LEV was evaluated using fluorescent imaging in mice bearing two tumor xenografts, DLD-1 and HT-29, models. Properties of the polymer conjugate containing BA-M-LEV and a NIR dye Dyomics782 (conjugate 7-dye) were compared with fluorescently labeled polymer precursor (conjugate 6dye) and similar micelle-forming polymer containing hydrophobic cholest-4-en-3-one instead of BA-M-LEV (conjugate 8-dye). We have found no release of the fluorescent polymer label within long term incubation in condition mimicking blood stream, thus all conjugates could be applicable in vivo.50


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In Figure 6 the overlay images of all mice 48 h after polymer application are arranged. Tumor accumulation was observed within all mice and both tumor types. Conjugate 8-dye gave the strongest signals for both experimental tumors whereas conjugate 6-dye showed lowest signal intensities for both tumors. Conjugate 7-dye containing BA-M-LEV showed similar accumulation in HT-29 carcinoma to that of conjugate 8-dye. Surprisingly, its accumulation in DLD-1 was not so substantial as the accumulation of conjugate 8-dye, but still obviously higher than that of conjugate 6-dye. We can conclude that polymer containing BA-M-LEV is accumulated within the solid tumor to higher extent than linear polymer, what could be ascribe to the self-assembly into the supramolecular micellar structures ensuring the solid tumor accumulation. Slightly lower accumulation in comparison to nondegradable micelle-forming polymer 8-dye is most probably connected to the degradability issue. Biodegradability of the polymer 7-dye leads to slight lowering of circulation time in blood, thus also slightly lower accumulation in solid tumor is observed. Inded, these preliminary observations demonstrated the high potential of micellar polymer-drug carriers with betulinic acid in the treatment of solid tumors. Further detailed study it is under way.


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Figure 6. Fluorescent images (800 nm) for all mice 48 h after application of polymer conjugates labeled by Dyomics782. Mice bearing two human colorectal cancer cell lines HT-29 (left side) and DLD-1 (right side) were used (n = 3 per group); tumors are marked with arrow. Ex vivo imaging After sacrifice, the selected organs and subcutaneous tumors were removed from one individual from each groups after 48 h and fluorescence was analyzed (see Figure 7). The fluorescence intensity in the tumor tissue correlated with the results shown in the previous section. Low signals from excised organs indicated low accumulation of polymer conjugates within these tissues as was also observed previously for similar HPMA copolymers.50 Only a higher concentration of the micellar conjugate 8-dye was observed in liver. Probably, it was caused by the stability of the micelles not ensuring their disassembly. However, this preliminary


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ex vivo study should be repeated with a larger amount of animals per group to do not jump to conclusions. Previous biodistribution study on cholesterol-based amphiphilic HPMA copolymers with doxorubicin showed no significiant drug accumulation in the liver tissue.39

Figure 7. Organ overlay fluorescent images (800 nm) 48 h after application of polymer conjugates labeled by Dyomics782. Mice bearing two human colorectal cancer cell lines HT-29 and DLD-1 was used (n = 1 per group).

Conclusion Various HPMA-based polymer conjugates with betulinic acid (BA) derivatives, designed for effective delivery to solid tumors due to passive tumor accumulation (EPR effect), were synthesized, and their properties were studied. The BA keto-derivatives differed in their structure and hydrophobicity (betulonic acid, BoA; BA levulinate, BA-LEV; BA 3-acetyl acrylate, BAAA and methylated BA levulinate, BA-M-LEV) were bound by pH-sensitive hydrazone bonds to the water-soluble hydrophilic linear polymer carriers. Due to the highly hydrophobic character of BA, its polymer conjugates formed micelles in aqueous solutions with an Rh of approximately 12


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– 14 nm when containing 3 – 4 mol% of BoA (conjugate 2d) or BA-LEV (conjugate 3c) or only 1 mol% of the more hydrophobic BA-M-LEV (conjugate 5a). A higher content of BA-derivative led to the formation of aggregates and prevented dissolution of the conjugate. The results of drug release experiments showed that both derivatives, BoA and BA-LEV, were quickly released from the micellar carrier at mildly acidic as well as at neutral pH (above 80 % within 8 h). The loss of the pH-dependent sensitivity of hydrazone bonds was explained by the presence of a free carboxyl group in the BA structure, which accelerates the hydrolysis of the hydrazone bond even at neutral pH. The use of a 3-acetyl acrylate spacer stabilized the hydrazone bond and thus prevented BA-AA release from conjugate 4 at these pH levels, thus documenting the unusability of this spacer for controlled BA release. However, the methylation of the carboxylic group of BA-LEV afforded pH-controlled release from the polymer conjugates at mild acid pH, mimicking the environment in tumor tissue or in the endosomes of tumor cells (90 % released within 8 h) and the sufficient stability of conjugate 5a at the neutral pH of blood (less than 10 % within 8 h). The drug release corresponded with the micelle structure disassembly. The in vitro formation of large particles ascribed to the aggregates of released BoA or BA-LEV was quite rapid, with a pH-independent growth rate. Because the toxicity of both conjugates 2d and 3c can be expected during their delivery in blood circulation due to fast drug release, they were assessed as not suitable for drug delivery. However, the disassembly of micelles formed from conjugate 5a was fast only at mild acid pH, mimicking the tumor microenvironment. The released hydrophilic polymer is expected to be eliminated from the organism by renal filtration without undesired accumulation in the body. The derivative BA-M-LEV itself and even conjugate 5a exhibited high cytotoxicity to selected tumor cell lines, comparable with that of free BA. The main benefit of the described drug delivery system for the BA-M-LEV derivative consists of its


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potential for tumor-specific delivery, in the form of self-assembled, high-molecular-weight structures. In vitro evaluation of conjugates and free drug shows that the derivate BA-M-LEV and even its polymer conjugate are keeping the cytotoxicity and are able to treat various cell lines. Moreover, we evaluated induction of cell death and apoptotic changes which are usually detected during the treatment with BA. Conjugates and BA-M-LEV showed different pattern of cell death/apoptosis induction in comparison with BA, however significantly higher than for cells treated with only polymer precursor (polymer 1) itself. These findings can point out applicability of these conjugates for in vivo application. The micellar structure improved the tumor accumulation of the polymer-drug conjugate as was shown using fluorescent imaging on mice bearing two xenograft tumors. Expectedly, the controlled drug release in the mild acid conditions of tumor tissue or cells will be followed by disassembly of the high-molecular-weight micellar structure into water-soluble short polymer fragments which should facilitate the removal of the polymer carrier from the body. These favorable properties of the conjugates provide a good basis for their further development and further in vivo studies of their anticancer activity.

Supporting Information Supporting information contains raw titration data of ITC, plots illustrating the disintegration of micellar conjugate with BoA or BA-LEV, and GPC chromatograms of polymer precursors. This material is available free of charge via the Internet at http://pubs.acs.org. *

Corresponding author. Tel.: +420-296 809 230; fax: +420-296 809 410, e-mail address:

[email protected] (Petr Chytil).


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Acknowledgments This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic within the National Sustainability Program I (Project POLYMAT LO1507) and National Sustainability Program II (Project BIOCEV-FAR), by the Czech Science Foundation (project No. 15-02986S) and by Russian Foundation for Basic Research (grant No.15-04-06664).

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