Development of biodegradable polyesters with various microstructures for highly controlled release of epirubicin and cyclophosphamide ˙ ołtowska, U. Piotrowska, E. Oledzka, U. Luchowska, M. Sobczak, K. Z´ A. Bocho-Janiszewska PII: DOI: Reference:

S0928-0987(16)30438-9 doi: 10.1016/j.ejps.2016.10.014 PHASCI 3760

To appear in: Received date: Revised date: Accepted date:

25 August 2016 4 October 2016 10 October 2016

˙ oltowska, K., Piotrowska, U., Oledzka, E., Luchowska, U., Please cite this article as: Z´ Sobczak, M., Bocho-Janiszewska, A., Development of biodegradable polyesters with various microstructures for highly controlled release of epirubicin and cyclophosphamide, (2016), doi: 10.1016/j.ejps.2016.10.014

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ACCEPTED MANUSCRIPT Development

of

biodegradable

polyesters

with

various

microstructures for highly controlled release of epirubicin and

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cyclophosphamide

K. Żółtowska a, U. Piotrowska a, E. Oledzka a, U. Luchowska a, M. Sobczak

Department of Biomaterials Chemistry, Chair of Inorganic and Analytical Chemistry,

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a

*, A. Bocho-Janiszewska b

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a,

Faculty of Pharmacy with the Laboratory Medicine Division, Medical University of Warsaw,

Chair of Chemistry, Department of Inorganic and Physical Chemistry, Faculty of Materials

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b

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1 Banacha St., Warsaw 02-097, Poland

Science and Design, Kazimierz Pulaski University of Technology and Humanities in Radom,

Corresponding author.

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*

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Chrobrego 27 St., Radom 26-600, Poland

E-mail addresses: [email protected] (K. Żółtowska), [email protected] (U. Piotrowska),

[email protected]

(E.

Luchowska),

[email protected]

or

Oledzka),

[email protected]

[email protected]

[email protected] (A. Bocho-Janiszewska).

1

(U.

(M. Sobczak),

ACCEPTED MANUSCRIPT ABSTRACT

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In this study, “predominantly isotactic”, disyndiotactic, and atactic polylactides (PLAs) and

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poly(ε-caprolactone)s (PCLs) were loaded with anticancer agents, epirubicin (EPI) and

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cyclophosphamide (CYCLOPHO), to investigate their properties as highly controlled delivery devices. It was found that the kinetic release of drugs from the obtained polyester matrices tested in vitro at 37°C and pH 7.4 was strongly dependent on average molecular weight (Mn)

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of the polymers as well as the PLAs’ microstructure. EPI and CYCLOPHO were released

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from various obtained matrices according to the diffusion, diffusion-degradation, and degradation mechanisms in a rather regular and continuous manner. Importantly, in some cases, the kinetics of the EPI and CYCLOPHO release was nearly zero-order, suggesting

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predominantly polymer degradation. It is shown that the drug release profiles can be tailored by a controlled design of the microstructure and Mn of polyesters, allowing use of the

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delivery systems.

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synthesized matrices for the development of highly controlled biodegradable anticancer drug

Keywords: Biodegradable and bioresorbable biomedical polyesters; Anticancer drug delivery systems; Controlled release; Epirubicin; Cyclophosphamide;

1. Introduction

In 2012, there were an estimated 14 million cases of cancer around the world. The majority of these deaths concerned lung, liver, stomach, colorectal, breast, and oesophageal cancers. Novel anticancer drug delivery systems (DDS) need to be obtained in order to

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ACCEPTED MANUSCRIPT substantially improve the efficiency of cancer therapy (Park et al., 2008). Various new types of anticancer drug carrier are available, such as liposomes, micelles, implants, microparticles,

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nanocapsules, and nanoparticles (Bajpai et al., 2008; Jagur-Grodzinski, 1999; Park et al.,

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2008). DDS should ideally deliver a drug to a specific site in a specific time and release

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pattern.

Epirubicin (EPI), the 4′-epimer of the anthracycline doxorubicin (DOX), is an anthracycline antineoplastic agent that inhibits DNA replication, transcription, and repair by

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binding to nucleic acids, which is commercially available for intravenous administration. EPI

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is an analogue of DOX and a safer alternative, with comparable clinical activities as well as reduced side effects at equivalent dose. EPI has a wide range of clinical applications in diverse types of malignancy, including acute leukaemia, breast cancer, gastric cancer, non-

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Hodgkin’s lymphomas, ovarian cancer, pancreatic cancer, small-cell lung cancer, and softtissue sarcomas (Laurence et al., 2006).

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Cyclophosphamide (CYCLOPHO) is an antineoplastic agent metabolized to active alkylating metabolites. CYCLOPHO is used in the treatment of chronic lymphocytic

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leukaemia, lymphomas, soft-tissue and osteogenic sarcomas, and solid tumours. It is given orally or intravenously. CYCLOPHO is inactive until metabolized by the liver (Laurence et al., 2006).

In modern cancer therapy, a large majority of patients require simultaneous administration of two or more anticancer drugs. Combinatorial therapy has remarkable potential to overcome the problems of conventional oncology treatments, since an enhanced therapeutic outcome can be obtained by co-administration of multiple bioactive molecules (Bonneterre et al., 2004; Hu et al., 2010; Saracchini et al., 2013). In recent years, the utilization of polymeric carriers of EPI or CYCLOPHO has gained considerable attention. Encapsulation of drug molecules within polymeric particles protects

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ACCEPTED MANUSCRIPT them from efflux transporters, whereas the small size of particles facilitates entry across the biological membrane. Biodegradable or bioresorbable aliphatic polyesters have been widely

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exploited for encapsulation of anticancer drugs. Drug release rates, size, and loading can be

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easily manipulated to provide further control over drug delivery. Use of these polyester

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systems has been approved by the FDA (Conte et al., 2000; Hoste et al., 2004; Khandare and Minko, 2006; Sobczak et al., 2007; Uhrich et al., 1999).

Numerous new EPI delivery systems have been investigated up to now. They include

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the following: EPI-loaded self-assembled cholesterol-conjugated carboxymethyl curdlan

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nanoparticles (Wang et al., 2007); polypeptide and polyethylene glycol (PEG)-block-poly(Lglutamic acid) (Zhang et al., 2015); PEG-EPI conjugates (Canal et al., 2010); copolymers raclactic and glycolide (Tariq et al., 2015); poly(N-(2-hydroxypropyl)methacrylamide)

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copolymer-EPI conjugates (Rihova, 2009; Yang et al., 2015); poly(styrene-co-maleic acid) (Angelova and Yordanov, 2014); dextran (Marquez et al., 2002); dendritic PEG (Pasut et al.,

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2005); human monoclonal antibodies (Takahashi et al., 1999); and poly(sialic acid) (Greco et al., 2013). CYCLOPHO delivery systems have also been studied. There are known non- or

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pegylated liposome-encapsulated (Saracchini et al., 2013), poly(D,L-lactide-co-glycolide) microsphere (Abedin et al., 2015), and polyethylenimine (Cameron and Shaver, 2011; Jeong et al., 2010; Mansour et al., 2010) carriers of CYCLOPHO. Studies seeking to discover new carriers of these drugs are also underway. The main problem with the obtained carriers is probably the lack of control in the release of the mentioned anticancer agents. Therefore, biodegradable polyesters with various microstructures seem to be one of the most interesting and promising polymer groups for application in anticancer drug delivery systems. The preparation of novel carriers of EPI and CYCLOPHO which are characterized by a highly controlled drug release profile is being demanded both by the pharmaceutical industry and by medical practitioners.

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ACCEPTED MANUSCRIPT In our previous papers, new and very effective diethylzinc/gallic acid (ZnEt2/GAc) and diethylzinc/propyl gallate (ZnEt2/PGAc) catalytic systems were used for the first time to

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synthesize and characterize various non-toxic biomedical polymeric matrices (Zoltowska et

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al., 2015a, 2015b). The present paper is the continuation of our previous work. The

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synthesized ZnEt2/GAc and ZnEt2/PGAc catalytic systems were used for the synthesis of the polymeric carriers with different microstructures to load anticancer agents. In our current work we prepared new biodegradable polyester drug delivery systems for high controlled

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release of EPI or CYCLOPHO. A significant novelty of our findings relies on using

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biodegradable polyester matrices with different microstructures as an efficient solution for the modification of anticancer drugs-release properties. In our research, we showed for the first time that the microstructure of the polymer matrices is a crucial factor and played an

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important role on the EPI or CYCLOPHO controlled release rate. We believe that the obtained polyester matrices, with well-defined microstructure, can

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be practically applied as “long-”, “medium-”, or “short-term” EPI and CYCLOPHO

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controlled delivery systems.

2. Materials and methods

2.1. Materials

rac-Lactide (3,6-dimethyl-1,4-dioxane-2,5-dione, 99%, rac-LA, Sigma-Aldrich, Co., Poznan, Poland) was purified by recrystallization from ethyl acetate solution and dried in a vacuum oven at room temperature. ε-Caprolactone (2-oxepanone, 99%, CL, Sigma-Aldrich, Co., Poznan, Poland) was dried with calcium hydride and distilled under argon atmosphere

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ACCEPTED MANUSCRIPT before use. Toluene (Sigma-Aldrich, Co., Poznan, Poland) was dried over potassium or phosphorus pentoxide. Epirubicin hydrochloride (4′-Epidoxorubicin hydrochloride, EPI, Co.,

Poznan,

Poland),

cyclophosphamide

(2-[bis(2-

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Sigma-Aldrich,

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chloroethyl)amino]tetrahydro-2H-1,3,2-oxazaphosphorine 2-oxide, CYCLOPHO, Sigma-

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Aldrich, Co., Poznan, Poland), diethylzinc (ZnEt2, solution 15 wt% in toluene, SigmaAldrich, Co., Poznan, Poland) and propyl gallate (3,4,5-trihydroxybenzoic acid propyl ester, ≥98%, PGAc, Sigma-Aldrich, Co., Poznan, Poland) were used as received from the

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manufacturer. Phosphate buffer solution (pH 7.4 ± 0.05, 0.1 M, PBS, potassium dihydrogen

Poland) was also used as received.

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phosphate/disodium hydrogen phosphate, 20°C, Avantor Performance Materials, Gliwice,

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2.2. Synthesis of polyester matrices

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A diethylzinc/propyl gallate (ZnEt2/PGAc) catalytic system was freshly prepared under argon atmosphere at room temperature immediately before reactions, according to our

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previously described method (Zoltowska et al., 2015a, 2015b). The ROP of CL or rac-LA was carried out in a glass tube in the presence of ZnEt2/PGAc as catalysts immediately before reactions, according to our procedure (Zoltowska et al., 2015a, 2015b). The 3 g of monomer and the required amount of ZnEt2/PGAc were placed in a 20 mL glass ampoule under argon atmosphere. The reaction vessel was then kept standing in a thermostated oil bath at 40-80 °C for 16 or 48 h (Tables 1 and 2). When the reaction time was completed, a cold reaction product was dissolved in CH2Cl2 and precipitated from cold methanol with diluted hydrochloric acid (5% aqueous solution). The organic phase was separated, washed with distilled water and dried to a constant weight.

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ACCEPTED MANUSCRIPT Spectroscopy data of PCLs 1

H-NMR (CDCl3, δ, ppm): 4.03 [2H, t, -CH2CH2CH2CH2CH2OC(O)-], 2.27 [2H, t, -

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CH2CH2CH2CH2CH2COO-], 1.61 [4H, m, -CH2CH2CH2CH2CH2COO-], 1.38 [2H, m, -

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CH2CH2CH2CH2CH2COO-]; 13

CH2CH2CH2CH2CH2COO-],

28.1

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C-NMR (CDCl3, δ, ppm): 173.3 [-C(O)O-], 63.9 [-CH2CH2CH2CH2CH2OC(O)-], 33.8 [[-CH2CH2CH2CH2CH2OC(O)-],

25.4

[-

CH2CH2CH2CH2CH2COO-], 24.4 [-CH2CH2CH2CH2CH2COO-];

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FT-IR (KBr, cm−1): 2944 (νasCH2), 2867 (νasCH3), 1724 (νC=O), 1244 (νC-O);

Spectroscopy data of PLAs

H-NMR (CDCl3, δ, ppm): 5.10–5.25 [1H, q, -CH(CH3)-], 4.38 [1H, q, -CH(CH3)OH, end

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group], 1.50–1.60 [3H, d, -CH3];

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1

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C-NMR (CDCl3, δ, ppm): 169.0-169.7 [-C(O)O-], 69.0-69.4 [-OC(O)CH(CH3)O-], 67.2 [-

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OC(O)CH(CH3)OH, end group], 20.6 [-OC(O)CH(CH3)OH, end group], 17.1 [OC(O)CH(CH3)O-];

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FT-IR Data (KBr, cm−1): 2997 (υasCH3), 2947 (υsCH3), 2882 (υCH), 1760 (υC=O), 1452 (δasCH3), 1348–1388 (δsCH3), 1368–1360 (δ1CH+δsCH3), 1315–1300 (δ2CH), 1270 (δCH + υCOC), 1215–1185 (υasCOC + rasCH3), 1130 (rasCH3), 1100–1090 (υsCOC), 1045 (υC-CH3), 960–950 (rCH3 + υCC), 875–860 (υC-COO), 760–740 (δC=O), 715–695 (γC=O), 515 (δ1CCH3 + δCCO), 415 (δCCO), 350 (δ2C-CH3 + δCOC), 300–295 (δCOC + δ2C-CH3), 240 (τCC);

2.3. Preparation of poly(ε-caprolactone) devices of epidoxorubicin and cyclophosphamide

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ACCEPTED MANUSCRIPT PCL was dissolved in CH2Cl2 in an argon atmosphere. After that, the EPI or CYCLOPHO were slowly added to the PCL solution. The content of the flask was vigorously

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stirred. The samples were later dried to a constant weight in vacuo at room temperature. The

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obtained powder was compressed for 10 min using a hydraulic press (Specac, London, UK) at

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98 kN, in the form of discs of 13 mm diameter and 1 mm thickness. The mean weight of the developed devices was 100 mg, corresponding to approx. 10 mg of drug. The polymer devices

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without drug were prepared equally.

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2.4. Preparation of polylactide devices of epidoxorubicin and cyclophosphamide

PLA was dissolved in CH2Cl2 in an argon atmosphere. After that, the EPI or

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CYCLOPHO were slowly added to the PLA solution. The content of the flask was vigorously stirred. The solution was evaporated at ambient temperature. Then, the films were dried under

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reduced pressure. Discs of approx. 13 mm diameter and 1 mm thickness were obtained. The mean weight of the developed devices was 100 mg, corresponding to approx. 10 mg of drug.

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The polymer devices without drug were prepared equally.

2.5. In vitro studies of epidoxorubicin and cyclophosphamide release from polymeric devices

The polymeric devices were immersed in 0.1 M PBS (100 ml) at 37ºC. The agitation speed was 50 rpm. The sample solutions were withdrawn for the analysis at selected time intervals and replaced with a new buffer solution. The quantity of CYCLOPHO was analysed according to the Poland Pharmacopeia (Ed. VIII, 2008). The concentration of EPI released was also determined according to the Poland Pharmacopeia (Ed. IX, 2011). The samples were

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ACCEPTED MANUSCRIPT prepared in triplicate. The same procedure was employed for the polymeric devices without

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drugs.

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2.6. Measurements

The intrinsic viscosity of the obtained PCLs and PLAs was determined in N,Ndimethylformamide (DMF) (at 30 °C) using a Stabinger Viscometer SVM 3000. The

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viscosity average molecular weight was calculated with the Mark-Houwink equation

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(Zoltowska et al., 2015a, 2015b).

The number-average molecular weight (Mn) and molar mass dispersity (PD) of the obtained polyesters were determined by gel permeation chromatography (GPC). The GPC

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instrument (GPC Max + TDA 305, Viscotek) was equipped with Jordi DVB Mixed Bed columns (one guard and two analytical) at 30°C in CH2Cl2 (HPLC grade, Sigma-Aldrich) and

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at a flow rate of 1 mL/min, with RI detection and calibration based on narrow PS standards (ReadyCal Set, Fluka). The results were processed with OmniSEC software (ver. 4.7.

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Houston, Texas, USA) (Zoltowska et al., 2015a, 2015b). MALDI-TOF mass spectra were performed in a linear mode using an ultrafleXtreme™ (Bruker Daltonics, Coventry, UK) mass spectrometer using a nitrogen gas laser and DCTB as a matrix. The PCL and PLA samples were dissolved in THF (5 mg/mL) and mixed with a solution of DCTB (Zoltowska et al., 2015a, 2015b). The polymerization products were characterized by means of 1H or

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C-NMR (using

Varian 300 MHz recorded, Palo Alto, CA, USA) in deuterated chloroform (CDCl3) at room temperature. FT-IR spectra (PerkinElmer, Waltham, MA, USA) were measured from KBr pellets.

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ACCEPTED MANUSCRIPT The morphological assessment of the samples was carried out with Scanning Electron Microscope FEI Quanta 250 FEG (FEI Inc., Eindhoven, The Netherlands). Samples were

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mounted on aluminium stubs with double-sided carbon tape. The tests in low vacuum mode

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untreated samples, avoiding typical coating of the artifacts.

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with a water-atmosphere pressure of 130 Pa were performed. This allows measurements of

High-performance liquid chromatography (HPLC) measurements were performed WATERS apparatus (pump 600, autosampler 717plus) with the use of RP18 column (5 μm).

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The mobile phase consisted of methanol and acetonitryl (70:30) at the flow 1 ml/min. EPI

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was monitored at 254 nm.

The degree of degradation was determined from the weight loss (WL) of the polymeric samples according to the equation:

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WL = [(W0 – Wdegr)/W0] x 100 (%) Where, WL – the weight loss

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W0 - the weight of dry polymer sample before degradation

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Wdegr – the weight of dry polymer sample after degradation

3. Results and discussion

Nine various PLAs and three various PCLs samples were obtained for the preparation of the devices loaded with EPI and CYCLOPHO (Tables 1 and 2). ZnEt2/PGAc was used as a new non-toxic catalytic system for their synthesis (Zoltowska et al., 2015a, 2015b). The PLA samples were characterized by a different microstructure (“predominantly isotactic”, disyndiotactic, or atactic) (Table 2, Fig. 1), determined by NMR spectra (Coudane et al., 1997; Kasperczyk, 1995; Kasperczyk and Bero, 2000; Zoltowska et al., 2015a). The MPLA-1

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ACCEPTED MANUSCRIPT and MPLA-4 samples were disyndiotactic PLA (p2 ≈ 1), the MPLA-2 and MPLA-5 samples

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“predominantly isotactic” PLA (p2 ≈ 0.5), and the MPLA-3 and MPLA-6 samples atactic.

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-----

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Table 1

Characterization of PCL matrices obtained in the presence of ZnEt2/PGAc catalytic system. -----

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Table 2

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Characterization of PLAs matrices obtained in the presence of ZnEt2/PGAc catalytic system. -----

Fig. 1. Scheme of homopolymerization of rac-LA in the presence of ZnEt2/PGAc catalytic

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system and microstructure of the obtained PLAs.

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-----

The in vitro kinetic release of EPI or CYCLOPHO from the synthesized devices was

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determined at pH 7.4, 37°C over about 12 weeks (Figs. 2–5). The ordinate of the plot was calculated based on the cumulative amount of drug released, with respect to its amount in the matrices.

----Fig. 2. Cumulative release of EPI and CYCLOPHO from the MPCL devices during 12 weeks (each point represents the mean ± SD of three points). -----

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ACCEPTED MANUSCRIPT It was found that many factors influence the release of EPI or CYCLOPHO from the obtained devices, but mainly the kinds of polyester and their Mn or microstructure.

MPCL-3-EPI,

MPCL-1-CYCLOPHO,

MPCL-2-CYCLOPHO,

and

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MPCL-2-EPI,

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The difference in the release rate observed for the PCL devices (MPCL-1-EPI,

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MPCL-3-CYCLOPHO) was attributed to the difference in the Mn of the PCL matrices (Fig. 2). The rate of in vitro drug release increased as the Mn of the matrices decreased. The percentage of the EPI released after 12 weeks of incubation was about 79% for MPCL-1-EPI

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(obtained from MPCL-1, Mn = 4 300 g/mol), 76% for MPCL-2-EPI (obtained from MPCL-

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2, Mn = 9 100 g/mol), and 71% for MPCL-3-EPI (obtained from MPCL-3, Mn = 17 500 g/mol). Similarly, 84% of CYCLOPHO was released from MPCL-1-CYCLOPHO, 80% from MPCL-2-CYCLOPHO, and 75% from MPCL-3-CYCLOPHO. Interestingly, the

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drugs were released in two phases from the MPCL-3 samples (phase I: nought to six weeks; phase II: after seven weeks). It was also found that EPI or CYCLOPHO were released in a

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rather regular and continuous way from the MPCL-3 matrices in phase II. Furthermore, CYCLOPHO was released more quickly than EPI. For instance, the percentage of EPI

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released from MPCL-1 was about 56%, 67%, 75%, and 79% after three, six, nine, and 12 weeks of incubation, whereas for CYCLOPHO these values were about 61%, 72%, 80%, and 84% after three, six, nine, and 12 weeks of incubation. The comparison of the PLA chain microstructure of the matrices loaded with EPI or CYCLOPHO showed that the drug release processes proceeded differently (Figs. 3–5). The results demonstrated that the release rate of drugs from the PLA matrices was mainly dependent on the polyester tacticity. The rate of EPI or CYCLOPHO release decreased as follows: PLA atactic (Fig. 3) > PLA disyndiotactic (Fig. 4) > PLA “predominantly isotactic” (Fig. 5). For example, approx. 77%, 50%, and 44% of EPI was released after 12 weeks from MPLA-6-EPI (obtained from the atactic PLA), MPLA-4-EPI (obtained from the

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ACCEPTED MANUSCRIPT disyndiotactic PLA) and MPLA-5-EPI (obtained from the “predominantly isotactic” PLA), respectively. The kinetic rates for CYCLOPHO release from the synthesized PLA matrices

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showed the same trends: 79%, 53%, and 45% of CYCLOPHO was released after 12 weeks

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from MPLA-6-CYCLOPHO (obtained from the atactic PLA), MPLA-4-CYCLOPHO

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(obtained from the disyndiotactic PLA), and MPLA-5-CYCLOPHO (obtained from the “predominantly isotactic” PLA), respectively. Furthermore, it was also observed that the rate of EPI or CYCLOPHO release depended on the Mn of PLA matrices. The amount of EPI

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released was about 52% for MPLA-2-EPI (obtained from MPLA-2, Mn = 9 200 g/mol) and

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44% for MPLA-5-EPI (obtained from MPLA-5, Mn = 16 800 g/mol) within 12 weeks. Similarly, 53% and 45% of CYCLOPHO was released from MPLA-2-CYCLOPHO and MPLA-5-CYCLOPHO, respectively. As shown, drugs were released from MPLA-6

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(obtained from the atactic PLA) and MPLA-1 (obtained from the disyndiotactic PLA) in two

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stages.

-----

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Fig. 3. Cumulative release of EPI and CYCLOPHO from the MPLA devices (obtained from the atactic PLA) during 12 weeks (each point represents the mean ± SD of three points). -----

Fig. 4. Cumulative release of EPI and CYCLOPHO from the MPLA devices (obtained from the disyndiotactic PLA) during 12 weeks (each point represents the mean ± SD of three points). ----Fig. 5. Cumulative release of EPI and CYCLOPHO from the MPLA devices (obtained from the “predominantly isotactic” PLA) during 12 weeks (each point represents the mean ± SD of three points).

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ACCEPTED MANUSCRIPT -----

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Three kinds of mechanism have already been proposed for drug release from the

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biodegradable matrices: Fickian diffusion through the polymer matrix, diffusion through

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pores in the matrix, and drug liberation by polymer erosion (Alexis, 2005; Dash et al., 2010; Kasperczyk and Bero, 2000; Siepmann and Gopferich, 2001). The release data points were subjected to zero-order and first-order kinetics and Higuchi and Korsmeyer–Peppas models to

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evaluate the kinetics and release mechanisms of the drug from the obtained polyester matrices

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(Tables 3–8). According to the Korsmeyer–Peppas model, for the diffusion-degradationcontrolled drug release system, the exponent value (n) varied between 0.45 and 0.89 (anomalous, non-Fickian). When n was close to 0.45, diffusion (Fickian diffusion) dominated

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in the process. When n was close to 0.89 (zero-order release), degradation controlled the release (Alexis, 2005; Dash et al., 2010; Kasperczyk and Bero, 2000; Siepmann and

----Table 3

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Gopferich, 2001).

Models of kinetic and drug release mechanism. -----

In our study, the release kinetics of EPI or CYCLOPHO from the PCL devices can be effectively described by the Higuchi and Korsmeyer–Peppas models (R2 > 0.97). It was established that exponent n of the Korsmeyer–Peppas model is characterized by a different drug release mechanism and its applied to the first 60% of drug released (Alexis, 2005; Dash

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coefficient R2 was chosen to assess the approximation accuracy.

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Table 4

Analysis data of EPI and CYCLOPHO release from PCL matrices.

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-----

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Table 4 shows that the exponent n in the Korsmeyer–Peppas model was approx. 0.45 for the MPCL-1 and MPCL-2 devices, providing further support for a diffusion-controlled release mechanism (Table 5). For the MPCL-3 devices, the analysis was carried out on data

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obtained from two drug release phases. The n value was 0.431 for MPCL-3-EPI and 0.394 for MPCL-3-CYCLOPHO in phase I, suggesting that drug release was governed mainly by

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Fickian diffusion. Interestingly enough, the n value in phase II was 0.948 for MPCL-3-EPI and 1.039 for MPCL-3-CYCLOPHO. Higher n values indicate that non-Fickian kinetics is

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operational. In phase II, the release exhibited a near-zero-order release profile (R2 was 0.9984 for MPCL-3-EPI and 0.9873 for MPCL-3-CYCLOPHO) (Table 4). We assume that water easily penetrated amorphous domains of PCL in phase I, and a diffusion period probably occurred. In phase II, EPI or CYCLOPHO were slowly released from the PCL matrices, as compared to phase I. Our results are logical due to the fact that the crystalline domains are relatively resistant to hydrolysis.

----Table 5 Summary of EPI and CYCLOPHO release kinetics from PCLs and transport mechanisms.

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ACCEPTED MANUSCRIPT -----

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The release curves for atactic PLAs exhibit a one- (MPLA-3-EPI, MPLA-3-

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CYCLOPHO) or two-phase release profile (MPLA-6-EPI, MPLA-6-CYCLOPHO) (Fig.

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3). The n values were 0.383 and 0.325 for MPLA-3-EPI and MPLA-3-CYCLOPHO, respectively (Tables 6 and 7), suggesting a Fickian diffusion-controlled mechanism. In the case of MPLA-6-EPI and MPLA-6-CYCLOPHO, the initial release level (phase I) is a

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more-or-less diffusion-controlled parabolic profile (Tables 6 and 7). The value ranged from

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0.368 to 0.393. The second stage (phase II, after six days) is likely a diffusion-degradationcontrolled mechanism; the n value was 0.631 and 0.612 for MPLA-6-EPI and MPLA-6-

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CYCLOPHO.

-----

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Table 6

----Table 7

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Analysis data of EPI release from PLA matrices.

Analysis data of CYCLOPHO release from PLA matrices. -----

The release curves for disyndiotactic PLA exhibit a one- (MPLA-4-EPI, MPLA-4CYCLOPHO) or two-stage release pattern (MPLA-1-EPI, MPLA-1-CYCLOPHO), similarly to the atactic PLA matrices (Fig. 4, Tables 6–8). For MPLA-4-EPI and MPLA-4CYCLOPHO, the n value was 1.017 and 0.971, respectively. This is likely due to a degradation-controlled mechanism. The release exhibited a near-zero-order release profile. R2

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ACCEPTED MANUSCRIPT was 0.9946 and 0.9869 for MPLA-4-EPI and MPAL-4-CYCLOPHO, respectively. For the MPLA-1 device, the analysis was carried out on data obtained from two phases of EPI or

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CYCLOPHO release. Phase II was from a four-day process. When the drugs’ release data

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were analysed using the Korsmeyer–Peppas model, n was 0.312 (MPLA-1-EPI) and 0.302

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(MPLA-1-CYCLOPHO) in phase I, suggesting that drug release was governed mainly by Fickian diffusion. In phase 2, the n value was 0.628 and 0.609 for MPLA-1-EPI and MPLA-

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1-CYCLOPHO, suggesting a diffusion-degradation-controlled mechanism.

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----Table 8

Summary of EPI and CYCLOPHO release kinetics from PLAs and transport mechanisms.

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D

-----

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The devices obtained from the “predominantly isotactic” PLA matrices show a more controlled release profile (Fig. 5, Tables 6–8). For the MPLA-2-EPI, MPLA-5-EPI, MPLA-

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2-CYCLOPHO and MPLA-5-CYCLOPHO samples, the n values ranged from 0.963 to 1.039. High R2 values (from 0.9815 to 0.9944) were obtained for the near-zero-order kinetics model. The R2 values of the Higuchi model were also high (from 0.9816 to 0.9856). The controlled drug release profiles were obtained with no significant burst release. This suggests that EPI and CYCLOPHO release from the “predominantly isotactic” PLA matrices is a highly controlled process. The lower drug release rate from MPLA-2 and MPLA-5 is a logical consequence of the increase in crystallinity due to the presence of isotactic blocks in the polymer chains. The hydrolytic degradation tests of the obtained PCL and PLA matrices were conducted under the same conditions as the drug release experiments. In vitro degradation of

17

ACCEPTED MANUSCRIPT the polyester matrices was controlled by the WL (Figs. 6 and 7), determined after three, six,

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nine, and 12 weeks of degradation.

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-----

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Fig. 6. The weight loss of PCL matrices during 12 weeks (each point represents the mean ± SD of three points). -----

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Fig. 7. The weight loss profiles of PLA matrices during 12 weeks (each point represents the

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mean ± SD of three points). -----

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The WL values for MPCL-1, MPCL-2, and MPCL-3 ranged from 57 to 63% after 12 weeks of degradation. The degradation process of PCL matrices was rather slow and for

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MPCL-1 and MPCL-2 non-regular. As mentioned above, drug release from MPCL-1 and MPCL-2 was governed by Fickian diffusion. In the case of MPCL-3, the matrix weight loss

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was regular (Fig. 6). This is consistent with the fact that drugs in phase II exhibited a nearzero-order release profile from MPCL-3 (Tables 4 and 5). The WL of PLA matrices ranged from 64 to 62 for MPLA-3 and MPLA-6 (obtained from the atactic PLA), from 50 to 40 for MPLA-1 and MPLA-4 (obtained from the disyndiotactic PLA), and from 42 to 35 for MPLA-2 and MPLA-5 (obtained from the “predominantly isotactic”) within 12 weeks (Figs. 6 and 7). It was found that the WL of PLAs increased as follows: matrices obtained from the PLA “predominantly isotactic” < matrices obtained from the PLA disyndiotactic < matrices obtained from the PLA atactic. The degradation of MPLA-4, MPLA-2, and MPLA-5 was rather regular. However, in the case of the MPLA-3, MPLA-6, and MPLA-1 samples, the weight loss was non-regular. These

18

ACCEPTED MANUSCRIPT results are comparable with the EPI and CYCLOPHO release results, due to the fact that drugs were released with near-zero-order kinetics from the MPLA-4, MPLA-2, and MPLA-5

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samples.

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Characterization of the surface of the PCL and PLA samples after degradation was

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also characterized byestablished using SEM analysis. As an example, the SEM images of MPCL-2 in the original state, as well as after six and nine weeks of degradation, are shown in Figs. 8(a-c). In comparison to the MPCL-2 matrix before degradation (Fig. 8a), the surface of

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the polymer after six and nine weeks of degradation exhibited well developed and severe

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cracking all over the surface, indicating significant oxidative and hydrolytic damage (Figs. 8b and 8c).

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-----

Fig. 8a. SEM micrographs of PCL matrice before degradation process.

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Fig. 8b. SEM micrographs of PCL matrice after 6 weeks’ degradation process.

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Fig. 8c. SEM micrographs of PCL matrice after 9 weeks’ degradation process. -----

4. Conclusions

This paper has examined biomedical “predominantly isotactic”, disyndiotactic or atactic polylactides (PLAs) and poly(ε-caprolactone)s (PCLs) as controlled-release carriers of anticancer epidoxorubicin (EPI) and cyclophosphamide (CYCLOPHO).

19

ACCEPTED MANUSCRIPT The most significant conclusions are as follows: 1. The rate of drugs release and matrix degradation increased as the number-average

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molecular weight of the obtained polyesters decreased.

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2. The rate of degradation of PLA matrices and drugs release was mainly dependent on the

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PLA microstructure, and increased as follows: “predominantly isotactic” PLA < disyndiotactic PLA < atactic PLA.

3. Importantly, in some cases, EPI and CYCLOPHO were released regularly and continuously

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from the obtained polyester devices.

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4. Anticancer EPI and CYCLOPHO were released:

a) from the PCL and atactic PLA matrices according to the diffusion and diffusiondegradation mechanisms (dependent on the Mn)

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b) from the disyndiotactic PLA matrices (also dependent on the Mn) according to the diffusion-degradation and degradation mechanisms; for the disyndiotactic PLA (Mn = 16 500

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g/mol), near-zero-order kinetics was observed; c) from the “predominantly isotactic” PLA matrices (Mn = 9 200 - 16 800 g/mol) according to

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the degradation mechanism; near-zero-order kinetics was also observed; 5. Importantly, in some cases, drug “burst release” was not observed during the degradation process (devices obtained from the “predominantly isotactic” PLA). We hope that the resulted biodegradable matrices (polycaprolactone and isotactic, disyndiotactic or atactic polylactides), which were synthesized in the presence of new, effective and non-toxic catalytic systems, are good potential candidates to be applied in the technology of various EPI or CYCLOPHO delivery carriers. Furthermore, we believe that our preliminary, but promising results, will let in the future to elaborate the technology of new high-controlled, short-, medium-, or long-term controlled EPI or CYCLOPHO delivery systems (mainly micro- and nanoparticles).

20

ACCEPTED MANUSCRIPT

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Acknowledgments

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This work was supported by the research programmes (Project Young Researcher FW23/PM32D/14: “Synthesis and analysis of polyurethane conjugates of cyclophosphamide ” and Mini-Grant Student FW23/NM2/15/15: "Innovative biodegradable carriers of cytostatics -

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synthesis and structural analysis") of the Medical University of Warsaw. The authors are

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indebted to Andrzej Plichta (Warsaw University of Technology) for the GPC measurements, Monika Pisklak and Violetta Kowalska (Medical University of Warsaw) for the spectroscopy

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measurements.

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References

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Polymers 7, 2410-2430.

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Zoltowska, K., Piotrowska, U., Oledzka, E., Sobczak, M., 2015a. Efficient

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diethylzinc/gallic acid and diethylzinc/gallic acid ester catalytic systems for the ring-opening polymerization of rac-lactide. Molecules 20, 21909-21923.

Zoltowska, K., Sobczak, M., Oledzka, E., 2015b. Novel zinc-catalytic systems for ring-

MA

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opening polymerization of epsilon-caprolactone. Molecules 20, 2816-2827.

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List of captions

Fig. 1. Scheme of homopolymerization of rac-LA in the presence of ZnEt2/PGAc catalytic

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system and microstructure of the obtained PLAs. Fig. 2. Cumulative release of EPI and CYCLOPHO from the MPCL devices during 12 weeks

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(each point represents the mean ± SD of three points). Fig. 3. Cumulative release of EPI and CYCLOPHO from the MPLA devices (obtained from the atactic PLA) during 12 weeks (each point represents the mean ± SD of three points). Fig. 4. Cumulative release of EPI and CYCLOPHO from the MPLA devices (obtained from the disyndiotactic PLA) during 12 weeks (each point represents the mean ± SD of three points). Fig. 5. Cumulative release of EPI and CYCLOPHO from the MPLA devices (obtained from the “predominantly isotactic” PLA) during 12 weeks (each point represents the mean ± SD of three points).

25

ACCEPTED MANUSCRIPT Fig. 6. The weight loss of PCL matrices during 12 weeks (each point represents the mean ± SD of three points).

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Fig. 7. The weight loss profiles of PLA matrices during 12 weeks (each point represents the

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mean ± SD of three points).

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Fig. 8a. SEM micrographs of PCL matrice before degradation process.

Fig. 8b. SEM micrographs of PCL matrice after 6 weeks’ degradation process.

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Fig. 8c. SEM micrographs of PCL matrice after 9 weeks’ degradation process.

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Table 1

Characterization of PCL matrices obtained in the presence of ZnEt2/PGAc catalytic system. Table 2

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Characterization of PLAs matrices obtained in the presence of ZnEt2/PGAc catalytic system. Table 3

Table 4

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Models of kinetic and drug release mechanism.

Table 5

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Analysis data of EPI and CYCLOPHO release from PCL matrices.

Summary of EPI and CYCLOPHO release kinetics from PCLs and transport mechanisms. Table 6 Analysis data of EPI release from PLA matrices. Table 7 Analysis data of CYCLOPHO release from PLA matrices. Table 8 Summary of EPI and CYCLOPHO release kinetics from PLAs and transport mechanisms.

26

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Figure 1

27

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Figure 2

28

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Figure 3

29

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Figure 4

30

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Figure 5

31

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Figure 6

32

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Figure 7

33

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Figure 8a

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34

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Figure 8b

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35

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Figure 8c

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ACCEPTED MANUSCRIPT Table 1 Characterization of PCL matrices obtained in the presence of ZnEt2/PGAc catalytic system. Molar ratio

Temp.

Mn a

PD a

[Zn]0 : [CL]0

(°C)

[Da]

MPCL-1

1/50

60

4 300

MPCL-2

1/100

60

9 100

MPCL-3

1/200

80

17 500

Mv c

(%)

[Da]

5

4 900

1.74

3

10 300

2.01

0

19 100

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T

No.

MC b

SC R

1.58

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Reaction conditions: reaction temperature - 60°C, reaction time - 48 h; determined by GPC (Mn corrected by a factor of ca. 0.47 (Zoltowska et al., 2015b));

b

MC (macrocyclic content) determined by MALDI TOF MS;

c

determined by viscosity method (K = 1.94·10−4 dL/g and α = 0.73) (Zoltowska et al.,

D

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a

AC

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2015b);

37

ACCEPTED MANUSCRIPT Table 2 Characterization of PLAs matrices obtained in the presence of ZnEt2/PGAc catalytic system. Mn a

(°C)

(h)

[Da]

MPLA-1

1/100

40

16

8900

MPLA-2

1/100

60

16

9200

MPLA-3

1/100

60

48

9600

MPLA-4

1/200

40

16

16500

MPLA-5

1/200

60

16

16800

MPLA-6

1/200

60

a

48

PD a (%) 1.38

Mvc p2

Li

T

8700

0.90

2.22

0

[Da]

2

1.57

3

9700

0.56

3.57

0

1.96

17

10100

-

-

0.49

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[Zn]0/[rac-LA]0

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No.

MC b

T

Time

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Temp.

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Molar Ratio

1.63

2

17700

0.92

2.17

0

1.87

2

17500

0.59

3.39

0

18200

2.28

15

17100

-

-

0.53

determined by GPC; Mn corrected by a factor of ca 0.58 (Zoltowska et al., 2015a); MC (macrocyclic content) determined by MALDI TOF MS;

c

determined by viscosity method (K = 2.21·10−4 dL/g and α = 0.77) (Zoltowska et al., 2015a);

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b

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p2 - coefficient of stereoselectivity calculated from the equation presented in (Coudane et al., 1997; Kasperczyk, 1995; Kasperczyk and Bero, 2000);

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T - transesterification coefficient (Coudane et al., 1997; Kasperczyk, 1995; Kasperczyk and Bero, 2000);

Li = 2/p2 - average length of lactyl units (Coudane et al., 1997; Kasperczyk, 1995; Kasperczyk and Bero, 2000).

38

ACCEPTED MANUSCRIPT Table 3 Models of kinetic and drug release mechanism. Equation

Zero order

T

Kinetics model

First order

log F  log F0 

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F  kt

Higuchi

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Korsmeyer–Peppas

kt 2.303

1 2

F  kt

F  ktn (F < 0.6)

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F - fraction of drug released up to time (t), F0 - initial concentration of drug, k - constant of

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the mathematical models, n - exponent of Korsmeyer–Peppas model.

39

ACCEPTED MANUSCRIPT Table 4 Analysis data of EPI and CYCLOPHO release from PCL matrices. First order

Higuchi

model

model

model

R2

R2

R2

MPCL-1-EPI

0.9076

0.9743

MPCL-2-EPI

0.9122

MPCL-3-EPI

-

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n

0.9814

0.9864

0.447

0.9712

0.9865

0.9973

0.454

-

-

-

-

0.9936

0.9899

0.431

0.9920

0.9967

0.9998

0.948

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R2

0.9637

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0.9508 (phase I) MPCL-3-EPI

D

0.9984

model

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MPCL-3-EPI

Korsmeyer-Peppas

T

Zero order No.

TE

(phase II)

0.9076

0.9809

0.9814

0.9806

0.396

MPCL-2- CYCLOPHO

0.8985

0.9707

0.9796

0.9890

0.403

MPCL-3- CYCLOPHO

-

-

-

-

-

0.9421

0.9563

0.9892

0.9856

0.394

0.9873

0.9807

0.9878

0.9804

1.039

CE P

MPCL-1-CYCLOPHO

AC

MPCL-3- CYCLOPHO (phase I)

MPCL-3- CYCLOPHO (phase II)

40

ACCEPTED MANUSCRIPT Table 5 Summary of EPI and CYCLOPHO release kinetics from PCLs and transport mechanisms. Release kinetics and transport mechanism

MPCL-1-EPI

Fickian diffusion

MPCL-2-EPI

Fickian diffusion

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IP

T

No.

diffusion-degradation

MPCL-3-EPI

Fickian diffusion

MPCL-3-EPI

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(phase I)

matrix degradation (near zero order kinetics)

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MPCL-3-EPI (phase II)

Fickian diffusion Fickian diffusion

TE

MPCL-2- CYCLOPHO

D

MPCL-1-CYCLOPHO

diffusion-degradation

MPCL-3- CYCLOPHO

Fickian diffusion

CE P

MPCL-3- CYCLOPHO

(phase I)

matrix degradation (near zero order kinetics)

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MPCL-3- CYCLOPHO (phase II)

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ACCEPTED MANUSCRIPT Table 6 Analysis data of EPI release from PLA matrices. Zero order

First order

Higuchi Korsmeyer-Peppas model

model

model

R2

R2

R2

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model

T

No.

n

0.9828

0.9795

0.383

-

-

-

0.9767

0.9945

0.393

0.9852

0.9826

0.631

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R2

atactic PLAs 0.9082

0.9764

MPLA-6-EPI

-

-

0.9128

0.9322

MA

MPLA-6-EPI (phase I) MPLA-6-EPI

0.9761

D

0.9944

-

-

-

-

-

0.8553

0.8708

0.9414

0.9830

0.312

0.9855

0.9950

0.9960

0.9969

0.628

0.9946

0.9964

0.9807

0.9866

1.017

AC

(phase I)

disyndiotactic PLAs

CE P

MPLA-1-EPI

TE

(phase II)

MPLA-1-EPI

NU

MPLA-3-EPI

MPLA-1-EPI (phase II)

MPLA-4-EPI

“predominantly isotactic” PLAs MPLA-2-EPI

0.9815

0.9874

0.9826

0.9851

1.009

MPLA-5-EPI

0.9916

0.9950

0.9818

0.9872

1.037

42

ACCEPTED MANUSCRIPT Table 7 Analysis data of CYCLOPHO release from PLA matrices. Zero order

First order

Higuchi Korsmeyer-Peppas model

model

model

R2

R2

R2

IP

model

T

No.

n

0.9853

0.325

-

-

-

0.9327

0.9767

0.9948

0.368

0.9582

0.9700

0.9825

0.612

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R2

atactic PLAs MPLA-30.9826

-

-

MA

MPLA-6CYCLOPHO

0.9128

TE

CYCLOPHO

0.9792

AC

(phase II)

CE P

(phase I)

CYCLOPHO

D

MPLA-6-

MPLA-6-

0.9834

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0.9153 CYCLOPHO

disyndiotactic PLAs

MPLA-1-

-

-

-

-

-

0.8472

0.8618

0.9392

0.9849

0.302

0.9850

0.9952

0.9959

0.9970

0.609

CYCLOPHO MPLA-1CYCLOPHO (phase I) MPLA-1CYCLOPHO (phase II)

43

ACCEPTED MANUSCRIPT MPLA-40.9940

0.9869

0.9850

0.9875

0.971

CYCLOPHO

T

“predominantly isotactic” PLAs

0.9817

0.9892

0.9944

0.9969

0.9856

MPLA-5-

0.9816

AC

CE P

TE

D

MA

NU

CYCLOPHO

SC R

CYCLOPHO

44

0.9883

0.963

0.9817

1.039

IP

MPLA-2-

ACCEPTED MANUSCRIPT Table 8 Summary of EPI and CYCLOPHO release kinetics from PLAs and transport mechanisms. Release kinetics and transport mechanism

T

No.

IP

atactic PLAs

Fickian diffusion

SC R

MPLA-3

diffusion-degradation

MPLA-6

Fickian diffusion

MPLA-6

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(phase I) MPLA-6

MA

diffusion-degradation

(phase II)

disyndiotactic PLAs

MPLA-1

D

Fickian diffusion

CE P

(phase I)

diffusion-degradation

TE

MPLA-1

MPLA-1

diffusion-degradation

AC

(phase II) MPLA-4

matrix degradation (near zero-order kinetics) “predominantly isotactic” PLAs

MPLA-2

matrix degradation (near zero-order kinetics)

MPLA-5

matrix degradation (near zero-order kinetics)

45

AC

CE P

TE

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

Graphical abstract

46