ATRP of Methacrylic Derivative of Camptothecin Initiated with PLA

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ATRP of Methacrylic Derivative of Camptothecin Initiated with PLA toward Three-Arm Star Block Copolymer Conjugates with Favorable Drug Release Andrzej Plichta,*,† Sebastian Kowalczyk,† Krzysztof Kamiński,† Monika Wasyłeczko,† Stanisław Więckowski,† Ewa Olędzka,‡ Grzegorz Nałęcz-Jawecki,§ Anna Zgadzaj,§ and Marcin Sobczak‡ †

Chair of Chemistry and Technology of Polymers, Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664 Warsaw, Poland ‡ Department of Biomaterials Chemistry, Chair of Inorganic and Analytical Chemistry, Faculty of Pharmacy with the Laboratory Medicine Division, and §Department of Environmental Health Science, Faculty of Pharmacy with the Laboratory Medicine Division, Medical University of Warsaw, 1 Banacha St., Warsaw 02-097, Poland S Supporting Information *

ABSTRACT: Three-arm polylactides (PLA) containing 0.2, 7.6, and 13% of D-lactic acid monomeric units were obtained and refunctionalized into ATRP macroinitiators via esterification of hydroxyl groups with 2-bromoisobutyryl bromide. These polymeric matrices underwent enzymatic degradation with various rates and revealed negative results on cytotoxicity and genotoxicity tests. Camptothecin (CPT), which is an anticancer active substance, was transformed into acrylic monomers; however, simple CPT acrylate was not able to polymerization whereas methacrylate with a linker was ready for FRP and ATRP. The latter monomer was used for ATRP initiated with various PLA macroinitiators in order to form block copolymer conjugates of CPT with high load of drug. Based on kinetic studies at various temperatures, it was found out that the polymerization stopped at certain monomer conversion because of the ceiling temperature. The content of CPT in these conjugates was estimated by means of 1H NMR, quadruple detection array GPC, and elemental analysis and was in the range 8.0−16.9 wt %. The products were morphologically heterogeneous, and the shapes and size of the nano-/microstructures were influenced by crystallinity of the PLA segment which was shown in AFM images. Terpolymer block conjugates consisting of addition PEGMA monomeric units were synthesized as well in order to increase hydrophilicity of the polymers and to protect a lactone ring in CPT structure. The studies on CPT release were carried out in vitro and revealed that the rate of CPT discharge was influenced by the structure of PLA and conjugate composition; however, it was near to zero-order kinetics. The analysis using the Korsmeyer−Peppas model suggests that drug release was governed rather according supercase II transport (n > 1) which shows that it is a highly controlled process.

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DNA replication and RNA transcription in cells.9 A large number of low molecular weight CPT and CPTD conjugates were synthesized and studied. For instance, the conjugate of CPT with a ligand bearing Pt(II) revealed higher activity against lung cancer and lower toxicity than CPT,10 whereas the amphiphilic drug−drug conjugation of hydrophobic CPT with hydrophilic floxuridine led to the formation of nanostructures stable in water which were more stable and less toxic than both components separately; therefore, they could keep an appropriate ratio of drugs and more efficiently induce the apoptosis of colorectal cancer cells.11 The other component of the implantable plate must be a support material that provides appropriate mechanical properties

INTRODUCTION Modern pharmaceutical therapy has become a target oriented treatment, which means that the medicine applied should be selectively delivered to or deposited in the specified organ or tissue in the body, e.g., affected by cancer. Drug delivery systems are intensively studied and developed; therefore, treatment efficiency can be improved whereas side effects are diminished.1−6 Resorbable implantation plate comprising the drug of directed cytotoxicity is one of the formulation types which can be inserted directly into the human body area altered by cancer cells in order to cure the patient.7 There is a range of cytotoxic drugs which could be used for such a system; however, camptothecin (CPT) and its derivatives (CPTD), i.e., topotecan, irinotecan, exatecan, and lurtotecan, are most studied medicines these days.8 Drugs belonging to this family are cytostatics which disturb the cell cycle, causing cell death or inhibit cell’s divisions and growth by inhibiting the action of topoisomerase I, which is necessary for © XXXX American Chemical Society

Received: June 23, 2017 Revised: August 9, 2017

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DOI: 10.1021/acs.macromol.7b01350 Macromolecules XXXX, XXX, XXX−XXX

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correlation of structure and morphology of the conjugates on CPT release profiles and mechanisms. Such a system potentially allows to introduce much more than one CPT molecule conjugated to the support polymer matrix. Moreover, the purpose of this study was to develop the perspective biodegradable carriers for the well-controlled release of CPT.

and constitutes the matrix for the drug; therefore, usually it is a bioresorbable (biodegradable) and biocompatible thermoplastic (co)polymer, such as polylactide (PLA), poly(lactide-coglycolide), poly(lactide-co-ε-caprolactone), etc.12,13 Although lPLA fulfills the above requirements, it might be too much rigid, brittle, and stable for some medical applications according to its semicrystalline structure. On the other hand, the introduction of some of D-lactic acid (LAc) monomeric units (mu) randomly distributed in PLA structure decreases the degree of crystallinity.14 The constituents of the implantable plate can form a physical mixture, but the conjugation of drug and polymer matrix is often profitable.15−20 In the case of CPT and CPTD conjugation with polymers, for instance with poly(ethylene glycol) (PEG) or poly(L-glutamic acid), may improve chemical stability of the drug, its solubility in water, and/or miscibility with matrix, and finally it affects the kinetics of drug release to the body and efficacy of the treatment.21−25 A majority of polymer−CPT conjugates present in the literature concerns the systems of one polymer chain coupled with one or two molecules of drug. Usually, the reactive hydroxyl group in the drug structure plays the role of initiating species for the formation of the polymer chain, or it is used for coupling process with ready oligomer chain via its end group(s) and some coupling agents (e.g., diisocyanate).21,26−31 Often homopolymers of lactide (LA) and its copolymers with other heterocyclic esters were successfully studied for that purpose.32,33 On the other hand, starlike macromolecular conjugates of CPT were prepared as well. It allowed to bond up to four drug molecules to the end of PEG-bpolycaprolactone arms.34 A higher content of CPT (and other drugs) was achieved applying the hyperbranched star copolymer structure.35,36 In both latter cases the kinetics of drug release profiles was not discussed, but it looks like nonzero-order kinetics. However, there are just a few papers showing a (block) copolymer system in which one type of monomeric unit or segment belongs to the support material and the other one to the prodrug. In such a case the drug derivative must contain the group able to polymerization or coupling; therefore, much more than several drug molecules may be introduced to the polymer conjugate.37,38 This strategy allows to increase the load of immobilized drug per molecule; thus, the carrier chain can be extended as well, which can improve the mechanical properties compared to oligomers. Zhang et al. synthesized polyphosphoester-conjugated CPT prodrug with disulfide linkage for potent reduction-triggered drug delivery, in which the CPT moiety was bonded to polyphosphate copolymer via “click” reaction.39 The same type of conjugation was applied in the case of synthesis of the CPT−polyoxetane block copolymer system.40 On the other hand, the disulfide derivative of CPT was conjugated to polymethacrylate backbone consisting thiol functions, which allowed formation of micelles followed by cross-linking. CPT was then released in buffer; however, the burst release or nonlinear release profiles were observed.41 The aim of this paper was the synthesis and characterization of three-arm star block copolymer conjugates of CPT with PLA segments, which could be used for preparation of anticancer implantable plate. The combination of ring-opening polymerization (ROP) of LA and ATRP of CPT (meth)acrylates seems to be the appropriate route to the target. Although the union of these two popular techniques is well-known in synthesis of block42 and graft43−45 copolymers, the authors believed that study on polymeric conjugates of CPT would bring the new knowledge on polymerization of CPT monomers as well as the

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EXPERIMENTAL SECTION

Materials. L,L-Lactide (l,l-LA) (Beckmann Kenko GmbH as well as Boehringer) and D,D-lactide (d,d-LA) (Beckmann Chemikalien) were transferred under nitrogen directly from an airtight bag just before polymerization without purification. CPT (98%, AKSci), mono-2(methacryloyloxy)ethyl succinate (≥95%, Sigma-Aldrich), 4-(dimethylamino)pyridine (DMAP) (99%, Fluka), 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDAC) (98%, Molekula), and poly(ethylene glycol) methyl ether methacrylate (PEGMA) (Mn 300 g mol−1, Sigma-Aldrich) were used as supplied. Acrylic acid (99%, SigmaAldrich) was distilled under diminished pressure just before usage. Sn(EH)2 (92−100%), 2-bromoisobutyryl bromide (98%), triethylamine (TEA) (99.8%), ascorbic acid (99%), tris(2-pyridylmethyl)amine (TPMA) (98%), N,N,N′,N″,N″-pentamethyldiethylenetriamine (PMDETA) (99%), anhydrous THF (99.9% SureSeal), and anhydrous 1,4-dioxane (99.8%) were purchased from Sigma-Aldrich and used without further purification. Ethyl acetate (≥98.5%, Sigma-Aldrich) and DMF (99%, Chempur) were dried over molecular sieves 4 Å (2 mm, Reag. Ph Eur, Merck). CuBr (99.9%, Aldrich) and CuCl (99.9% Aldrich) were washed with glacial acetic acid (p.p.a., POCh), absolute ethanol (POCh), and diethyl ether (p.p.a., POCh), dried, and stored under vacuum in a desiccator. Trimethylolpropane (TMP) (97%, Aldrich) was dried under diminished pressure at 50 °C for 1 h before use. Dichloromethane (DCM) (HPLC grade, Aldrich) as a solvent for esterification was dried under reflux with CaH2, distilled on molecular sieves 4 Å, and stored under nitrogen or used without purification as an eluent for GPC. DCM (technical grade, POCh) and methanol (technical grade, POCh) for precipitation of polymers were used without purification. Activated basic aluminum oxide (Sigma-Aldrich), activated neutral aluminum oxide (Sigma-Aldrich), activated charcoal (Sigma-Aldrich), sodium hydrogen carbonate (pure, Chempur), and hydrochloric acid (38 wt %, POCh) were used for purification of polymers without specific treatment. Polymerization of LA (Exemplified by PLA-2). In a two-necked round-bottom flask equipped with a stirring bar, condenser, and gas bladder, 233 g (1.62 mol) of l,l-LA, 14.6 g (0.12 mol) of d,d-LA, 1.15 g (8.57 mmol) of TMP, and 0.14 mL (0.430 mmol) of Sn(EH)2 were placed under a nitrogen atmosphere. The flask has been located in the molten Wood’s alloy at 190 °C for 2 h, then the sample for monomer conversion evaluation was collected, the system was cooled down, and the product was dissolved in 700 mL of DCM and precipitated in 7 L of cold methanol. The product was filtered off, washed with methanol, and dried in a vacuum oven at 40 °C for 48 h. Esterification of PLA-OH with α-Bromoisobutyryl Bromide (Exemplified by PLA-2Br). In a three-necked round-bottom flask equipped with a stirring bar, cooling condenser, gas bladder, and dropping funnel, 100 g of PLA-2 was placed under a nitrogen atmosphere and dissolved in 360 mL of dry DMC. Then 13.1 mL (94.1 mmol) of TEA was added, and 11.1 mL (94.1 mmol) of αbromoisobutyryl bromide was introduced dropwise to cooled solution (0 °C). The reaction was carried out for 72 h at room temperature, and then 6 spoons of activated carbon was introduced to the system. After 24 h the solid was filtered off, and the filtrate was washed once with 200 mL of 0.1 M HCl and three times with 200 mL of 5 wt % NaHCO3. Finally, the organic phase was dried overnight with MgSO4 and then precipitated in a 3 L of cold methanol. The product was filtered off, washed with methanol, and dried in a vacuum oven at 40 °C for 48 h. After that, the product was mechanically crumbled into small pieces and washed vigorously for 1 h with 1 L of cold methanol in a beaker equipped with a mechanical stirrer in order to remove some residual low molar mass derivatives of α-bromoisobutyric acid. The final product was filtered off and dried in a vacuum oven at 40 °C for 48 h. B

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Macromolecules Synthesis of CPT Mono-2-(methacryloyloxy)ethyl Succinate (CPTMA). In a two-necked round-bottom flask equipped with stirring bar and cooling condenser with gas bladder, 11.0 g (57.4 mmol) of EDAC, 500 mL of dry DCM, and 11.1 mL (57.4 mmol) of mono-2(methacryloyloxy)ethyl succinate were placed and mixed under nitrogen. The mixture was cooled down to 0 °C, and 10.0 g (28.7 mmol) of CPT and 7.01 g (57.4 mmol) of DMAP were added. A greenish-yellow heterogeneous mixture was stirred at 0 °C for half an hour and then kept at room temperature overnight which resulted in a brown clear solution. The reaction mixture was washed three times with 350 mL of 1 M HCl, three times with 350 mL of 1 wt % NaHCO3, and once with 350 mL of brine. Finally, the organic phase dried overnight with 3 spoons of MgSO4 in the fridge. Then the filtrate was evaporated on rotavap, yielding the yellow crude product which was recrystallized from the mixture of methanol and DCM (95:5 v/v) obtaining yellow crystals in the form of fine needles of the yield of 86%. Synthesis of Starlike Block Co-/Terpolymer Conjugates via ATRP (Exemplified by Entries 2 and 5 from Table 2). In a Schlenk flask equipped with a stirring bar, 0.500 g (0.89 mmol) of CPTMA, 255 μL (0.89 mmol) of PEGMA (entry 5 only), 378 mg (12.5 μmol) of PLA1Br, 19.8 mL of dry THF, and 2.6 μL (12.5 μmol) of PMDETA (0.1 mL of stock solution in THF) were placed under nitrogen with a view to avoid a moisture, whereas in order to remove an oxygen, three freeze− thaw cycles were applied. After that, 1.24 mg (12.5 μmol) of CuCl was introduced to the system in a protective atmosphere. The mixture was placed in an oil bath at 60 °C, and after 4 h 47 μL (9.4 μmol, entry 2) or 20 μL (4.0 μmol, entry 5) of 0.2 M solution of ascorbic acid in DMF was introduced. Reactions were stopped after 27 h (entry 2) or 26 h (entry 5). The final reaction mixture was dissolved with 35 mL of DCM, passed through the neutral alumina column, followed by 0.2 μm PTFE syringe filter, and then precipitated with 500 mL of cold methanol, filtered off, and dried under vacuum at 40 °C for 24 h. Enzymatic Degradation Tests. Films used in the enzymatic degradation test were prepared by hot pressing at 170 °C under the pressure of 5−7 MPa for 90 s in the air in a hydraulic press and cut into 1.0 × 1.0 cm sheets with thickness in the range of 0.10−0.15 mm. Each sheet was placed in a separate vial containing 5 mL of Tris-HCl buffer (pH 8.6), 0.1 mg of proteinase K (Sigma-Aldrich, lyophilized powder, ≥30 units mg−1 of protein), and 1 mg of sodium azide (Sigma-Aldrich). Three replicate sheets in separate vials were used to determine the sheet weight loss at specified incubation time. The sheet-enzyme incubations were carried out at 37 °C in a rotary shaker (150 rpm). After a predetermined time, respective sheets were removed from the shaker incubator, rinsed thoroughly with distilled water, and then dried under reduced pressure (0.5 mbar) at 40 °C for 48 h. Experimental weight loss values and molar mass loss values represent averages of determinations from the three replicate films. In Vitro Studies of Drug Release. The polymeric carriers containing CPT were immersed in 0.1 M PBS at 37 °C. All experiments were carried out in triplicate. The agitation speed was 50 rpm. The sample solutions were withdrawn (using the filter) for the analysis at selected time intervals and replaced with a new buffer solution. The absorption of buffer solution was determined by a UV−vis spectrophotometer at the absorbance peak with a wavelength at 355 nm (lactone form).32 The absorbance peak correlated very well with the concentration of CPT. A linear calibration curve was obtained by measuring the absorption of solutions with predetermined CPT concentrations. The CPT release data points were subjected to a zero-order, a first-order, and Korsmeyer−Peppas models to evaluate the kinetics and the drug release mechanism from the obtained carriers.46,47 AFM Imaging. A Multimode 8 Nanoscope atomic force microscope (AFM, Bruker, U.S.A.) was used to image the samples surface. Silicon cantilevers with a spring constant of ca. 0.5 N m−1 HQ:NCS19/No Al (MIKROMASCH, Bulgaria) were applied for imaging in PeakForce tapping force microscopy mode. Calibration of the microscope was achieved by the imaging of calibration gratings supplied by the manufacturer. The images presented in this work are height or peak force error maps. The examination of surfaces for artifacts by AFM, and the reproducibility was performed in the common way, i.e., by changing the AFM cantilever and moving the sample in the X or Y direction or by

varying the scanning angle and scan rate. Data were analyzed with the dedicated Nanoscope 8.15 software. Other Measurements. 1H NMR analysis was performed on a Varian Mercury 400 MHz (for PLA), 500 MHz (for CPT-monomers), or 800 MHz (for conjugates) spectrometer using CDCl3 as a solvent. The molar mass and molar mass distribution were determined by GPC on a Viscotek system comprising GPCmax and TDA 305 [triple detection array (TDA): RI, IV, LS] equipped with DVB Jordi gel column(s) (102−107, linear, mixed bed) in DCM as an eluent at 30 °C at a flow rate of 1.0 mL min−1. TDA-GPC and QDA-GPC (comprising additional UV-PDA detector) were used for determination of molar mass, ĐM, and composition (QDA) of obtained PLA MIs and conjugates, respectively, based on absolute calibration method. The DSC measurements were performed using a TA Instruments DSC Q200 V24.2 Build 107 apparatus. One heating run from 20 to 200 °C was performed at a heating rate of 5 °C min−1. TGA measurements were conducted using a TA Instruments SDT Q600 instrument, at heating rate of 20 °C min−1 under a nitrogen atmosphere. Specific rotation measurements were carried out with an Optical Activity PolAAr 32 polarimeter using sodium lamp of λ = 589 nm and measurement tube of the length d = 100 mm at the sample concentration of 10 g L−1 in chloroform at 25 °C.

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RESULTS AND DISCUSSION Synthesis and Characterization of PLA Matrices. Three PLA matrices were prepared as macroinitiators of ATRP processes in order to obtain various block copolymers comprising of CPT species. Solely l,l-LA or combined with 7% and 12% d,d-LA was used for bulk ROP carried out at 190 °C for 2 h in the presence of catalytic amount of tin(II) 2ethylhexanoate and initiated with trimethylolpropane yielding 3-arms star-shaped PLA polymers with high monomer conversions (95−96%). Afterward, each hydroxyl-terminated arm was converted into ATRP initiation species via esterification with 2-bromoisobutyryl bromide (Scheme 1). The structures of

Scheme 1. Synthesis of PLA-Br Macroinitiators of ATRP

hydroxyl- and bromo-terminated polymers were confirmed with H NMR spectra, which proved that LA polymerization was initiated mostly with TMP, and then the end groups were quantitatively refunctionalized into bromoesters (Figure 1S). Structural properties of PLA MIs such as Mn and ĐM were obtained from TDA-GPC analysis employing value of dn (dc)−1 = 0.0362 mL g−1, which was determined for PLA at 30 °C in DCM, whereas content of d-mu was based on polarimetric measurements. The results are shown in Table 1. The values of Mn of hydroxyl-terminated PLA were a bit lower than calculated ones, which means that some chains were linear (initiation with products of LA hydrolysis) or macrocyclic (intramolecular tranesterification). However, ATRP MIs showed narrower distributions and higher molar masses because of subsequent fractionation through precipitation processes during purification 1

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Macromolecules Table 1. Results on Synthesis of PLA Macroinititorsa entryb

αLAc (%)

Mn,cald (kg mol−1)

Mne (kg mol−1)

ĐM e

d,d-LAf (%)

d-LAc mug (%)

PLA-1 PLA-1Br PLA-2 PLA-2Br PLA-3 PLA-3Br

95 n/a 95 n/a 96 n/a

27.8 n/a 27.7 n/a 27.9 n/a

24.7 30.2 19.3 57.1 21.0 36.7

1.41 1.31 1.61 1.19 2.05 1.39

0 n/a 7 n/a 12 n/a

0.2 1.0 7.6 7.0 13 12

K enzyme in TRIS·HCl buffer at the body temperature of 37 °C for 7 days. Weight loss (panels A and B) and the evolution of Mn (panel C) of the samples prepared in the form of thin squared sheets (melt pressed) were examined, and the results are shown in Figure 1. Disintegration of hydroxyl-terminated PLA was

Conditions of ROP of LA: bulk, 190 °C, 2 h, [LA]0:[TMP]0: [Sn(EH)2]0 = 201:1:0.05. bEntries with “Br” suffix relate to the bromo-terminated PLA, whereas other lines relate to hydroxyl PLA intermediates. cConversion of LA based on 1H NMR spectra. dMn,cal = 28.97αLA + 0.13 kg mol−1. eMeasured with TDA-GPC using dn (dc)−1PLA = 0.0362 mL g−1. fMolar % of d,d-LA used for ROP in the mixture with l,l-LA. gMolar % of d-LAc mu in the polymer estimated with polarimetry. a

(Figure S2). Three-arm PLA MIs were synthesized in order to increase the load of CPT acrylic monomer which could be introduced into the one molecule of block copolymer comparing to linear ones at quite low degree of polymerization of methacrylic segment. Some d-mu (7 or 12%) were involved with the aim of disruption of regular microstructure and reduce crystallization degree. On the other hand, the polymerization of l,l-LA led to some minute amount of d-mu in the polymer structure as well, caused by racmiazation processes. Despite the fact that CPT shows a specific and strong cytostatic properties, the other components of therapeutic system should not be toxic; therefore, the PLA matrices were tested for their toxicity against cells and genes. The umu test is a bioassay for evaluating the genotoxic potential of environmental samples and chemical compounds. The test organism is Salmonella typhimurium TA1535/pSK1002. As a response to different types of DNA damage, the umuC gene in bacterial cells is induced which is a part of the SOS system. The test strain is genetically modifiedthe umuC gene activity is linked to the synthesis of enzyme, which converts colorless substrate into the yellow product which can be quantified colorimetrically at 420 nm. Additionally, the bacteria growth (G) is evaluated by a measurement of an optical density to determine the cytotoxicity of tested samples. The genotoxic potential of sample is presented as the induction ratio (IR)the mentioned enzyme activity ratio of tested sample in comparison to the negative control. Samples with IR ≥ 1.5 are considered as genotoxic.48 The results of the umu test for the highest concentration (0.66 mg mL−1) of aqueous extracts from PLA polymers revealed that none of tested samples were cytotoxic for the Salmonella typhimurium (G > 0.5) (Table S1). The intensity of bacteria growth in comparison with the negative control was not inhibited, but some of the highest concentrations of extracts slightly stimulated it. Moreover, none of tested extracts increased the IR over 1.5; therefore, none of them exhibited genotoxic potential (IR < 1.5). The pH of the extracts of all tested polymers after 24 h of incubation was in the range 6.0−7.5. These values of pH had no effect on toxicity. In the Spirotox test the samples did not cause any negative effects for the testing organism. Moreover, in the Microtox the PE dropped below 20%; thus, we can consider that the samples of all tested polymers were nontoxic for bacteria (Table S2). The PLA part of the copolymer implant should undergo a slow degradation in the body; therefore, the obtained matrices were studied for in vitro enzymatic degradation employing Proteinase

Figure 1. Enzymatic degradation profiles of PLA stars: weight loss of hydroxyl-terminated PLA stars (A) and bromine-terminated PLA MIs (B) as well as evolution of Mn change (C). Standard deviations for panels A and B are given in Tables S3 and S4 of the Supporting Information.

faster than in the case of ATRP MI, which suggests that hydroxyl end group may be involved in the mechanism of degradation. On the other hand, the samples composed of almost only l-LAc mu degraded slower than those enriched in some d-LAc mu due to the possible crystallization of “pure” l-LAc forms. The evolution of Mn (panel C) in the case of hydroxyl-terminated polymers showed that after first 24 h of degradation the parameter increased significantly because some fractions of low molar mass were washed out or decomposed quite fast, whereas these kinds of chains were removed from PLA-Br MIs previously, during D

DOI: 10.1021/acs.macromol.7b01350 Macromolecules XXXX, XXX, XXX−XXX

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Radical polymerization tests were carried out and they indicated that sterically hindered CPT-A with bulky drug molecule closely bonded to acrylic function was not able to FRP or ATRP under a range of conditions, whereas CPTMA which contains an aliphatic spacer between CPT and methacrylic acid molecule underwent controlled and uncontrolled radical polymerization in THF, DMF, ethyl acetate, and 1,4-dioxane at 60−70 °C. It is worth to mention that FRP and ATRP of CPTMA acted as precipitation polymerization yielding for instance a polymer of molar mass Mn (and ĐM) 0.78 (1.99) or 11.6 kg mol−1 (1.04) measured with GPC and calculated with conventional calibration (PS-GPC) or absolute calibration (TDA-GPC, using determined value of dn (dc)−1 = 0.1692 mL g−1), respectively. It suggests that separation of CPTMA polymer on DVB gel column is strongly affected by interaction of analyte and bed. Thermogravimetric analysis of PCPTMA carried out in nitrogen revealed two-step pyrolytic decomposition with maxima at 291 °C (19.6%) and 422 °C (71.2%); thus, 28.8% of solid residue remained in the sample at 800 °C. ATRP Conjugation of CPTMA on PLA Matrices. Branched block copolymer conjugates were prepared by (co)polymerization of CPTMA (and PEGMA) initiated with PLA MIs under ATRP conditions (Scheme 3). The molar ratio of CPTMA to MI of 72:1 was applied in all systems (24 molecules of monomer per arm) and in the case of PEGMA usage both monomers were added in equimolar amount. The latter monomer was introduce into some systems in order to increase hydrophilicity of the final product and to mitigate of risk of lactone ring cleavage in CPT structure. The CuCl/PMDETA complex was implemented as a catalytic system toward high initiation efficiency through halogen exchange. Conditions and some results are listed in Table 2. Generally, the processes of CPTMA polymerization were carried out in THF at 60 °C. Kinetic data in the form of semilogarithmic plots acquired with UV-GPC (at λ = 390 nm, signals of monomer and polymer were well separated) for several

multiprecipitation purification steps. Therefore, in the case of MIs Mn was almost constant (PLA-1Br) or slightly decreased for less crystalline (PLA-2Br) or amorphous (PLA-3Br) products. It suggests that the degradation proceeded rather on the surface of the sample than in the bulk; however, probably in the case of the less crystalline structures some processes shall follow shallowly in bulk as well. Thermal stability of PLA stars under inter gas increased after transformation into bromo-MI because one can expect that the process of decomposition begins via depolymerization if possible. For instance, the temperatures of beginning and maximum rate of decomposition raised for PLA-1Br are 70 and 85 °C, respectively, compared to PLA-1 (Figure S3). Synthesis and Polymerizability of Acrylic Derivatives of CPT. Two acrylic monomers consisting of CPT moiety were prepared via nonequilibrium (Steglich type) esterification of tertiary hydroxyl group in the CPT molecule. For the synthesis of CPT acrylate (CPT-A) acrylic acid was used, whereas CPTMA was obtained with monoester derivative of succinic acid with the 2-(methacryloyloxy)ethyl group in the presence of EDAC and DMAP (Scheme 2). 1H NMR (Figure S3 for CPTMA) and 13C NMR spectra of both monomers conformed to their structure. Scheme 2. Synthesis of CPTMA

Scheme 3. Synthesis of Block Copolymer Conjugates of PLA and CPTa

a

CPTMA monomer was used in every reaction, whereas PEGMA was used optionally. Graphical representation of conjugates with unexteded arm, coupled and regular, is shown. E

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Macromolecules Table 2. Conditions and Results on Polymerization of CPTMA and PEGMA Initiated with Various PLA MIsa entry f

1

2 3 4 5 6 7 8 9

MI PLA-1Br

PLA-2Br PLA-3Br

temp (°C) 40 →60 60 80 90 60 60 60 60 60

time (h) 27 1 27 23 27 26 27 24 26 24

molar ratio of MI/CPTMA/ PEGMA

max conv of CPTMA (%)b

1:72:0

43 39 22 15 16 39 22 44 15 42

1:72:0 1:72:0 1:72:0 1:72:72 1:72:0 1:72:72 1:72:0 1:72:72

Mn,calc (kg/mol)c 48.9 47.1 39.3 36.5 36.9 66.0 42.8

Mn,GPC (kg/mol)d,e g

117.0 (107.0) 95.5 86.9 (81.8) 91.7 (89.4) 60.5 (59.9) 68.9 69.8 (67.0) 76.0 64.3 (63.6) 47.2

ĐM e g

g

2.20 (1.24)g 1.33 1.51 (1.20) 1.27 (1.17) 1.27 (1.15) 1.29 1.57 (1.18) 1.20 4.49 (1.14) 1.81

a

Reactions carried out in dry THF (entries 1, 2, and 5−9), DMF (3), or 1,4-dioxane (4); molar ratio of CPTMA:MI:CuCl:PMDETA was 72:1:1:1 (in entry 4 TPMA was used instead of PMDETA), where MI was three-arm PLA, PEGMA was used (entries 5, 7, and 9) as a comonomer in equimolar amount to CPTMA, solution of ascorbic acid in DMF was added after several hours in order to regenerate activators at the ratio of 3:1 with respect to MI, [CPTMA]0 = 40 ± 5 mmol L−1 based on solubility of MI and CPTMA. bCPTMA conversion was calculated by means of GPC with UV detector at 390 nm. cCalculated Mn = [CPTMA]0([MI]0)−1αCPTMAMCPTMA + MMI, where [CPTMA]0 and [MI]0 are initial concentration of monomer and MI, respectively, αCPTMA and MCPTMA are conversion and molar mass of monomer, whereas MMI stands for molar mass of MI; it was not calculated for entries 5, 7, and 9 because conversion of PEGMA was not analyzed. dResults from QDA-GPC (entries 1−4, 5, and 8) with UV detector working at 275 nm or TDA-GPC for other entries. eThe values without the parentheses relate to full copolymer distribution including coupling products, whereas the values in parentheses are calculated only for major copolymer distribution. fReaction carried out for 27 h at 40 °C and then heated up to 60 °C and carried out for 1 h. gSample was not precipitated.

detectors used for TDA-GPC comprised UV-PDA detector (λ working range: 190−500 nm). QDA-GPC is a powerful tool which allows to analyze appropriately not only molar masses of the copolymers but also the total composition and the distribution of each mu among fractions of molar masses. In this method it is necessary to know for calculations the specific refractive index increments (dn (dc)−1) and the relative extinction coefficients (dA (dc)−1 which is specific extinction coefficient proportional to the value for PS arbitrarily equaled to 10) of each mu The values of dn (dc)−1 are given for PLA and PCPTMA in this text above, whereas the dA (dc)−1 values determined at 275 nm were 2 and 142, respectively. The QDAGPC traces of copolymers are overlaid with TDA-GPC traces of respective PLA MIs and shown in Figure 4. The major distributions of copolymers are nicely shifted toward higher molar mases; however, a tiny “bump” at high molar mass is present, indicating some minute coupling of stars, although methacrylates terminate mainly via disproportionation. The combination of polymer stars produced high values of ĐM and slightly elevated values of Mn when calculated for full range of products, though if only major population of branched block copolymer was integrated the values of ĐM were quite low ( 0.89 the model corresponds to supercase II transport.50

both samples. It shows that the presence of PEG short chains in some way promotes the process. In Vitro Study on CPT Release. The in vitro kinetic studies on release of CPT from the synthesized conjugates were determined at pH 7.4 and 37 °C for 42 days (Figure 7). The ordinate of the plot was calculated based on the cumulative amount of drug released with respect to its initial amount in the conjugates. It was found out that many factors influenced the release of CPT; however, the predominant factors were the content of d-LAc mu in the PLA carriers and the composition of conjugates in the meaning of CPTMA and PEGMA weight fractions. The obtained data points of drug release were subjected to zero-order and first-order kinetics as well as Korsmeyer−Peppas models to evaluate the kinetics and release mechanisms of the CPT from the co- and terpolymer conjugates

M t (Ma)−1 = Kt n

(1)

where Mt(Ma)−1 is a fraction of released drug at time t, K is constant incorporating structural and geometric dosage form, and n is the release exponent indicating the drug release mechanism. It was observed that the kinetic of drug release is influenced by CPTMA mu content in the conjugates. For example, H

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Macromolecules Table 3. Composition (wt % of mu) of Polymer Conjugates of CPT Estimated with Various Analytical Techniques 1

entry

a

PLA

H NMR analysis PEGMA

QDA-GPC CPTMA

CPTMA

CPT

CPT per moleculeb

42.7d 22.7 27.1 23.7 22.6 12.8 18.5 21.3 24.4

26.5d 14.1 16.9 14.7 14.1 8.0 11.5 13.2 15.2

72.7d 33.1 43.2 25.3 27.8 15.3 25.1 24.2 20.5

c

1 2 3 4 5 6 7 8 9

elemental analysis CPTMA

61.3d 78.8 75.7 76.5 60.7 87.8 71.4 81.6 59.8

12.6 9.8 13.4

22.8 35.6d 22.6 18.5 18.2 n/a 12.9 n/a 15.6 n/a

38.7d 21.2 24.3 23.5 26.7 12.2 18.8 18.4 26.8

Entries correspond with entries in Table 2. bAverage number of CPT molecules per 1 molecule of conjugate, calculated as Mn,conjωCPTMA(560.55)−1. c Data obtained from crude sample (no precipitation was done) after 27 h at 40 °C. dData obtained from precipitated sample after an additional 1 h of heating at 60 °C with respect to the line above. a

Figure 5. 1H NMR spectrum of block terpolymer conjugate of PLA with CPTMA and PEGMA (entry 5, Table 2).

released in two phases (phase I: 0−14 days; interphase; phase II: >16 days). In phase I the active substance was released with rather first-order kinetics (R2 = 0.9656), whereas in phase II the process was rather regular and continuously followed zero order model (R2 = 0.9895, Table S6). On the other hand, the difference in the release rate observed for the conjugates was attributed to the various content of d-LAc mu in the PLA chain as well. Surprisingly, the higher molar percent of d-LAc mu the lower rate of drug release, e.g., 76.1%, 56.4%, and 36.1% of CPT was realeased after 42 days of incubation when PLA segment consisted 0.2% (PLA-1Br, entry 2), 7.6% (PLA-2Br, entry 6), and 13% (PLA-3Br, entry 8) d-LAc mu (Figure 7B). According to AFM study, one can suggest that in the case of entry 2 the ability to crystallization of PLA domains forces formation of nanostructures with much higher surface area than that for entry 8 which showed microstructures; therefore, the contact area of CPTMA segment with the buffer solution is higher for entry 2, causing faster drug release for that sample. Contrary to entry 2, in the case of entries 6 and 8 CPT was released from the conjugates in one phase with near-zero-order kinetics (R2 was 0.9942 and 0.9948, respectively). The kinetic profiles of drug release from starlike terpolymers composed of the blocks consisting CPTMA and PEGMA mu and PLA segments comprising of various d-LAc mu content are

Figure 6. AFM images of solvent-cast thin films of samples of entry 2 (panels A, B) and entry 8 (panels C−E). Images in panels A and C were analyzed in height mode whereas the others in peak force error mode. Entries refer to Table 2.

approximately 76.1%, 66.0%, and 54.2% of CPT were released after 42 days from conjugates comprising of PLA-1 segment and 22% (entry 2), 16% (entry 4), and 15% (entry 3) of CPTMA mu, respectively (Figure 7A). The CPT release for entries 3 and 4 followed near-zero order kinetics (R2 was 0.9944 and 0.9943, respectively). Interestingly, in the case of entry 2 the CPT was I

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Figure 7. CPT release profiles: (A) block copolymers of PLA-1Br and CPTMA obtained at various temperatures; (B) block copolymers of CPTMA and various PLA MIs; (C) block terpolymers of CPTMA, PEGMA, and various PLA MIs. (D-1) Weight loss of selected samples and (D-2) a ratio of weight loss of CPT to weight loss of sample. Entries refer to Table 2.

shown in Figure 7C (entries 5, 7, and 9). It seems that the presence of PEGMA mu affected the mechanism of release as well as it caused inversion of the order of release rates with respect to d-LAc mu content in PLA segment comparing to copolymers. The percentage of CPT released after 42 days of incubation from block terpolymers was 95.3% (entry 9), 76.6% (entry 7), and 73.9% (entry 5) for conjugates obtained from PLA-3Br, PLA-2Br, and PLA-1Br MI, respectively. In the case of entry 5 the CPT was released in two phases (phase I: 0−16 days; phase II: ≥17 days). It was a similar phenomenon to the case of copolymer (entry 2) comprising of the same MI (PLA-1Br), which contained mainly l-LAc mu Moreover, the first-order kinetics (R2 = 0.9973) and near-zero-order kinetics (R2 = 0.9796) were observed in phases I and II; accordingly, for entry 5 whereas only one phase of first-order kinetics was indicated in the case of entries 7 and 9. The analysis of the CPT release data using the Korsmeyer− Peppas model suggested that all the samples excluding entry 9 (n = 0.799) were governed rather according supercase II transport (n > 1). The entries 3, 4, 6, and 8 were characterized by the wellcontrolled drug release. The weight loss of these samples was rather regular and linear in time, and after 42 days of incubation it reached 49.9%, 59.8%, 52.5%, and 33.6% for entries 3, 4, 6, and 8, respectively (Figure 7D-1). The CPT was released without significant burst release, but evenly with the carrier degradation, so the ratios of the fraction of weight loss caused by CPT release to the total weight loss of sample were almost constant in time (Figure 7D-2). As the CPT was released from mentioned samples in a slow and very regular way, it can suggest that the CPT release from the synthesized conjugates is a highly

controlled process (near-zero-order kinetics) according to the degradation mechanism.

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CONCLUSIONS All synthesized three-arm-star PLA matrices before and after refunctionalization into ATRP MI were not cytotoxic or genotoxic, and their ability to crystallization and enzymatic degradation was d-LAc mu content dependent. Out of two obtained CPT-based monomers only one derivative with a spacer between a drug molecule and methacrylic moiety was ready to polymerization whereas CPTA did not form a polymer chain. However, CPTMA indicated a ceiling temperature; therefore, the polymerization kinetic could not attributed to the first order with respect to the monomer, and moreover lower monomer conversions were observed at elevated temperatures. The star-shaped block co- and terpolymers comprising of PLA block of various d-LAc mu content and methacrylic segment composed of CPTMA mu, and optionally PEGMA mu were obtained. Contrary to many similar systems described in the literature in this case much more than one drug molecule was bond to the PLA chain (arm). The composition was calculated based on 1H NMR; however, QDA-GPC was successfully implemented in order to follow distribution and total amount of CPT along molar mass fractions. These data were quite consistent with elemental analysis. AFM images of the surface of copolymer conjugates differed by d-LAc mu content shown diverse morphology and size of the domains, i.e., nanorods in the case of l-PLA and microporous for 12% d-LAc mu. The nanoobjects revealed higher surface area than the micro ones; therefore, cumulative CPT release after 42 days of in vitro J

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(4) Jahangirian, H.; Lemraski, E. G.; Webster, T. J.; RafieeMoghaddam, R.; Abdollahi, Y. A review of drug delivery systems based on nanotechnology and green chemistry: green nanomedicine. Int. J. Nanomed. 2017, 12, 2957−2978. (5) Maciejowska, J.; Kasperczyk, J.; Dobrzynski, P.; Bero, M. The influence of chain microstructure on hydrolytic degradation of glycolide/lactide copolymers used in drug delivery systems. J. Controlled Release 2006, 116, E6−E8. (6) Singh, V.; Tiwari, M. Structure-Processing-Property Relationship of Poly(Glycolic Acid) for Drug Delivery Systems 1: Synthesis and Catalysis. Int. J. Polym. Sci. 2010, 2010, ID 652719.110.1155/2010/ 652719. (7) Brunton, L. L.; Chabner, B.; Knollmann, B. C. The Pharmacological Basis of Therapeutics, 12th ed.; Goodman & Gilman: 2011. (8) Liu, Y. Q.; Li, W. Q.; Morris-Natschke, S. L.; Qian, K. D.; Yang, L.; Zhu, G. X.; Wu, X. B.; Chen, A. L.; Zhang, S. Y.; Nan, X.; Lee, K. H. Perspectives on Biologically Active Camptothecin Derivatives. Med. Res. Rev. 2015, 35, 753−789. (9) Wu, S. F.; Hsieh, P. W.; Wu, C. C.; Lee, C. L.; Chen, S. L.; Lu, C. Y.; Wu, T. S.; Chang, F. R.; Wu, Y. C. Camptothecinoids from the seeds of Taiwanese Nothapodytes foetida. Molecules 2008, 13, 1361−1371. (10) Cincinelli, R.; Musso, L.; Dallavalle, S.; Artali, R.; Tinelli, S.; Colangelo, D.; Zunino, F.; De Cesare, M.; Beretta, G. L.; Zaffaroni, N. Design, modeling, synthesis and biological activity evaluation of camptothecin-linked platinum anticancer agents. Eur. J. Med. Chem. 2013, 63, 387−400. (11) Hu, M. X.; Huang, P.; Wang, Y.; Su, Y.; Zhou, L. Z.; Zhu, X. Y.; Yan, D. Y. Synergistic Combination Chemotherapy of Camptothecin and Floxuridine through Self-Assembly of Amphiphilic Drug-Drug Conjugate. Bioconjugate Chem. 2015, 26, 2497−2506. (12) Khandare, J.; Minko, T. Polymer-drug conjugates: Progress in polymeric prodrugs. Prog. Polym. Sci. 2006, 31, 359−397. (13) Nair, L. S.; Laurencin, C. T. Biodegradable polymers as biomaterials. Prog. Polym. Sci. 2007, 32, 762−798. (14) Rafael Auras, L.-T. L., Selke, S. E. M.; Tsuji, H. Poly(lactic acid): Synthesis, Structures, Properties, Processing, and Applications, 1st ed.; John Wiley & Sons, Inc.: Hoboken, NJ, 2010; p 528. (15) Garnett, M. C. Targeted drug conjugates: principles and progress. Adv. Drug Delivery Rev. 2001, 53, 171−216. (16) Uhrich, K. E.; Cannizzaro, S. M.; Langer, R. S.; Shakesheff, K. M. Polymeric systems for controlled drug release. Chem. Rev. 1999, 99, 3181−3198. (17) Pasut, G.; Veronese, F. M. Polymer-drug conjugation, recent achievements and general strategies. Prog. Polym. Sci. 2007, 32, 933− 961. (18) Hu, X.; Tian, J.; Liu, T.; Zhang, G.; Liu, S. Photo-triggered release of caged camptothecin prodrugs from dually responsive shell crosslinked micelles. Macromolecules 2013, 46, 6243−6256. (19) Huang, W.; Wang, Y.; Zhang, S.; Huang, L.; Hua, D.; Zhu, X. A facile approach for controlled modification of chitosan under γ-ray irradiation for drug delivery. Macromolecules 2013, 46, 814−818. (20) Ossipov, D.; Kootala, S.; Yi, Z.; Yang, X.; Hilborn, J. Orthogonal chemoselective assembly of hyaluronic acid networks and nanogels for drug delivery. Macromolecules 2013, 46, 4105−4113. (21) Fan, N. Q.; Duan, K. R.; Wang, C. Y.; Liu, S. Y.; Luo, S. F.; Yu, J. H.; Huang, J.; Li, Y. P.; Wang, D. X. Fabrication of nanomicelle with enhanced solubility and stability of camptothecin based on alpha,betapoly[(N-carboxybutyl)-L-aspartamide]-camptothecin conjugate. Colloids Surf., B 2010, 75, 543−549. (22) Haverstick, K.; Fleming, A.; Saltzman, W. M. Conjugation to increase treatment volume during local therapy: a case study with PEGylated camptothecin. Bioconjugate Chem. 2007, 18, 2115−2121. (23) Minko, T.; Paranjpe, P. V.; Qiu, B.; Lalloo, A.; Won, R.; Stein, S.; Sinko, P. J. Enhancing the anticancer efficacy of camptothecin using biotinylated poly(ethyleneglycol) conjugates in sensitive and multidrugresistant human ovarian carcinoma cells. Cancer Chemother. Pharmacol. 2002, 50, 143−150. (24) Singer, J. W.; Bhatt, R.; Tulinsky, J.; Buhler, K. R.; Heasley, E.; Klein, P.; de Vries, P. Water-soluble poly-(L-glutamic acid)-Gly-

incubation was higher as well in that case. The release rates of the CPT were shown to be directly dependent on the kind of the polymeric matrices and increased as follows: PLA-3Br < PLA2Br < PLA-1Br in the case of copolymers, whereas it was opposite when PEGMA was introduced to the conjugate. Importantly, in some cases, the CPT was released on a regular basis and continuously from the synthesized polymeric conjugates. It is also worth to notice that the CPT was released according to the degradation mechanism (near-zero-order kinetics was observed). We hope that our promising results will let in the future to elaborate the technology of new highcontrolled, long- or medium-term CPT delivery systems.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.7b01350. A part of the Experimental Section; additional figures (e.g., 1 H NMR spectra, GPC traces, TGA thermograms) and tables (e.g., data on PLA matrices toxicity, DSC data, CPT release models fitting) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(A.P.) E-mail [email protected], phone +48 22 234 5632. ORCID

Andrzej Plichta: 0000-0002-4935-2627 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Science Center, Poland (grant 2013/09/B/ST5/03480). ABBREVIATIONS CPT, camptothecin; CPTA, CPT acrylate; CPTMA, CPT mono-2-(methacryloyloxy)ethyl succinate; DCM, dichloromethane; DMAP, 4-(dimethylamino)pyridine; EDAC, 1-ethyl-3(3-(dimethylamino)propyl)carbodiimide hydrochloride; LA, lactide; LAc, lactic acid; MI(s), macroinitiator(s); m.u., monomeric units; PEGMA, poly(ethylene glycol) methyl ether methacrylate; PLA, polylactide (hydroxyl-terminated; PLA-Br, polylactide (bromine-terminated); PMDETA, N,N,N′,N″,N″pentamethyldiethylenetriamine; PS-, conventional calibration (narrow polystyrene standards); QDA-, quadruple detection array; ROP, ring-opening polymerization; Sn(EH)2, tin(II) 2ethylhaxanoate; TDA-, triple detection array; TMP, trimethylolpropane; TPMA, tris(2-pyridylmethyl)amine.

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L

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