Optimized Synthesis of Poly(deoxyribose) Isobutyrate, a Viscous

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Biological and Medical Applications of Materials and Interfaces

Optimized synthesis of poly(deoxyribose) isobutyrate (PDIB), a viscous biomaterial for BMP-2 delivery Farid Mirmohseni, Tegan L. Cheng, Farshad Oveissi, Mohammadreza Behi, Aaron Schindeler, David G. Little, Sina Naficy, Fariba Dehghani, and Peter Valtchev ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b20126 • Publication Date (Web): 27 Dec 2018 Downloaded from http://pubs.acs.org on January 10, 2019

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ACS Applied Materials & Interfaces

Optimized synthesis of poly(deoxyribose) isobutyrate (PDIB), a viscous biomaterial for BMP-2 delivery Farid Mirmohseni1, Tegan Cheng 2, Farshad Oveissi1, Mohammadreza Behi1, Aaron Schindeler1,2,3, David Little 2,3 , Sina Naficy1, Fariba Dehghani1, §, Peter Valtchev1, §,* 1 School

of Chemical and Biomolecular Engineering, The University of Sydney, Sydney, 2006,

Australia 2 Orthopaedic

Research & Biotechnology, The Children’s Hospital at Westmead, Westmead,

NSW 2145, Australia 3 Paediatrics

& Child Health, The Children’s Hospital at Westmead, Westmead, NSW 2145,

Australia §: Equal

*:

senior authors

Corresponding Author

KEYWORDS: Condensation polymerisation, deoxyribose, esterification, poly deoxyribose isobutyrate anhydride, bone tissue engineering

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ABSTRACT

Injectable and phase transitioning carriers from natural polysaccharides have great potential for the minimally-invasive delivery of therapeutic proteins in the field of bone tissue engineering. In this study, a novel and highly viscous drug carrier was synthesised by a sequential process of deoxyribose polycondensation and esterification. The effect of synthesis parameters on the molecular weight, viscosity and adhesion of the material was studied and correlated to temperature of polycondensation (Tp and tp), time and temperature of esterification (Te and te), and the molar ratio of the monomer (R). The formulations were evaluated for molecular weight and distribution using properties using Gel permeation chromatography (GPC), chemical structure by Fourier transform infrared (FTIR) and NMR Spectra, and rheological properties using rheometer. Formulations illustrated a wide range of viscosities (0.736 to 2225 Pa s), adhesion (0.896 to 58.45 N) and molecular weight (637 to 4216 Da), where viscosity was significantly reduced in the presence of low amounts of solvents (10-20%). The sustained release of Bovine Serum Albumin (BSA) was observed over 42 days in vitro. The biocompatibility of PDIB as well as its potential as a BMP delivery system was assessed in vivo using a rat ectopic bone model, where bone nodules were observed at 2 weeks. In summary, PDIB is a promising molecule with multiple applications for protein delivery, including for bone tissue engineering.

1. INTRODUCTION Over the past few decades, the pharmaceutical industry has been rapidly growing with over 200 therapeutic protein and peptides already approved, where over 50% have been approved just in the past five years.1 Therapeutic proteins, either native or synthesised, have revolutionised the


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healthcare sector by providing various task-specific drugs.2 based on engineered peptides, enzymes, antibodies, hormones and cytokines for therapy of immune disorders,3 cancers,4 infections,3 chronic wounds,5 bone repair,6 and other diseases.7 In particular, recombinant bone morphogenetic proteins (BMPs) are potent bone forming agents used to promote open fracture repair, critical defect healing, and spine fusion in orthopaedics. Currently delivered via an acellular collagen sponge with poor biomechanical properties and burst release kinetics, there is significant attention on identifying superior carriers for delivery. The options for delivery of therapeutic proteins in a sustained manner and without any loss in bioactivity are still limited due to the fragile nature of proteins. A major drawback is limited in vivo stability of proteins caused by degradation and proteolytic cleavage, which leads to their removal from the blood system. Therefore, repetitive administrations of these drugs are required through intravenous or subcutaneous injections, leading to patient discomfort. To address these issues, other non-invasive delivery methods including oral, nasal, pulmonary, and transdermal have been proposed, but none have been fully successful because of enzymatic degradation and poor solubility of proteins, and their nonlinear pharmacokinetics.8-9 In the context of BMPs, high viscosity sugar-based carriers such as sucrose acetate isobutyrate (SAIB) have shown favourable properties in a range of preclinical orthopaedic models.10-11 However, SAIB is a hydrophobic liquid that shows poor solubility with BMP-2 and other hydrophilic compounds and proteins and such agents typically form a colloid suspension. To surmount these shortcomings, much attention has been devoted to finding new means for delivering protein-based therapeutics with greater efficiency and prolonged biological activity. These research efforts have followed two primary pathways: the first path has focused on increasing the half-life of the therapeutic molecules through chemical modification, for example 3 Environment ACS Paragon Plus


by amino acid substitution,12 protein acylation 13 or PEGylation,14 to optimise the pharmacokinetic properties of the macromolecules. The second path has primarily aimed at developing minimallyinvasive delivering methods. Ideally, a suitable carrier should prolong the release and retain the protein activity in vivo by protecting the proteins from antibody neutralization. In this regard, polymers are excellent candidates as their physical and chemical properties can be easily modified. Some methods have been previously reported on the development of delivery systems based on hydrogels,15 nanoparticles16 and liposomes17 via drug entrapment or encapsulation. These methods, however, have shown numerous drawbacks. For instance, hydrogels have a large initial burst release due to diffusion-controlled release, microsphere-based system generated acidic microenvironment during bioerosion, and lipid-based systems have low encapsulation efficiency and poor release kinetics.18 Polysaccharide-based polymers such as those based on chitosan are non-toxic, biocompatible and the most abundant organic compounds found on the planet.19 Due to the presence of multiple functional groups on their backbones such as carboxylic, hydroxyl, amine and aldehyde, systems based on polysaccharides have tailorable physicochemical properties making them suitable for drug delivery applications.20 Previous studies have conﬁrmed that the injectable hydrogels prepared by natural polysaccharides show better biocompatibility than many synthetic polymers such as chitosan, alginate and dextran

18.

For practical applications, efforts have been made to

modify these natural polymers through their functional groups via esterification and etherification, enabling them to readily dissolve in aqueous or organic solvents. Previous studies have also shown that the chemical modification of polysaccharides, such as collagen, can increase adhesion and proliferation of various cell types.21 Deoxyribose is an endogenous sugar that fulfils important biological roles in mammals. Being a precursor to DNA, deoxyribose (dR) is formed by the 4 Environment ACS Paragon Plus

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phosphorolytic degradation of deoxynucleosides, catalysed by thymidine phosphorylase (TP).22 Deoxyribose is also a pro-angiogenic molecule that has shown to stimulate endothelial cell migration in various in vitro and in vivo studies.23

Here, we present a detailed study on synthesis and characterisation of deoxyribose-based in situ forming polymeric carriers. Aside from minimally invasive administration procedures, these polymeric systems can be simply manufactured and provide sustained drug release, due to higher retention at the injection site as a highly viscous material. Once injected, the polymeric solutions (in ethanol) form a viscous and adhesive depot, due to the diffusion of the organic solvent, which can act as structural function and a carrier vehicle for bioactive compounds. The synthesis of deoxyribose polymers was carried out through a sequential sugar polycondensation and esterification process. We designed and developed a controlled reactor system to monitor and control a variety of reaction parameters, particularly temperature, during the highly exothermic esterification reaction. To widen the application of the synthesised deoxyribose polymers, flow characteristics of the various formulations were fully explored and then correlated to the reaction condition, the reaction yield, and the molecular weight. Moreover, we correlated the adhesion results with rheological behaviour and processing parameters of each formulation. Synthesized PDIB was able to solubilize hydrophilic or hydrophobic drugs, making it suitable for delivery of bioactive proteins such as BMP-2. Recombinant bone morphogenetic protein-2 (BMP-2) was delivered by PDIB in a rat ectopic bone formation model as an injectable carrier, where nodules were observed and examined at 2 weeks by tissue histology and microCT analysis.



2. EXPERIMENTAL SECTION 2.1. Materials Deoxyribose (2-deoxy-D-ribose) was received from Carbosynth (Berkshire, UK). Sodium acetate, citric acid, isobutyric anhydride, Bradford reagent and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (Missouri, USA) and used as received. BMP-2 was purchased as part of the INFUSETM bone graft kit from Medtronic Australasia (North Ryde, Australia). 2.2. Synthesis of poly(deoxyribose isobutyrate) Poly(deoxyribose isobutyrate) (PDIB) was synthesised by step-growth polymerisation of deoxyribose, followed by esterification using isobutyric anhydride and sodium acetate as catalysts. 24

Briefly, deoxyribose (16 g) was added to acetic acid (4 ml) and citric acid (80 mg) in a jacketed

reactor (Atlas, UK). The mixture was then stirred at 250 rpm and fixed temperatures, from 90 to 110 °C, for different polycondensation times ranging from 10 to 40 min. Conventionally, this polycondensation reaction involves heating the carbohydrate until it is completely melted in the presence of a catalyst such as phosphoric acid. Poor control over the outcome of melt polycondensation increases the chance of carbohydrate degradation, which leads to the formation of impurities such as furfural and heterocyclic compounds.25 To overcome this issue, we conducted polycondensation reaction in a solution of glacial acetic acid, rather than in melt. Acetic acid was chosen as it is not only a suitable solvent for deoxyribose but also increases mass and heat transfer within the solution and subsequently prevents local overheating. The polycondensed deoxyribose was then esterified by adding a certain volume of pre-heated isobutyric anhydride, varied from 30 to 119 ml, to the reactor. The addition of isobutyric anhydride was in a dropwise manner to prevent


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overshooting of temperature. Afterwards, sodium acetate (1.4 g) was added to the reactor as a catalyst, and the reaction continued for another 10 to 20 min. The product was then purified by adding ethanol (120 ml) at 80 °C over 30 min followed by cooling down to 70°C. This procedure aimed to convert the remaining low volatile isobutyric anhydrides (TBoiling = 182°C) to a more volatile compound, i.e. ethyl isobutyrate (TBoiling =112 °C). The purification continued by washing the product with ethanol and water, followed by centrifugation at 6000 rpm for 3 min. The precipitate was oven dried at 50 °C for 72 h under vacuum. The formation of ester bond was verified via 1H-NMR (Varian, 400MR) using methanol (CD3OD) as solvent. 2.3. Experimental design Orthogonal design of experiments was applied using IBM SPSS (Version 22) to investigate the effect of reaction parameters on physicochemical properties of PDIB such as viscosity, adhesiveness and molecular weight. The tested parameters were time (tp) and temperature (Tp) of polycondensation, time (te) and temperature of esterification (Te), and the initial molar ratio of deoxyribose to isobutyric anhydride (R). Table 1 shows the designed experimental conditions for this study.



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Table 1. Orthogonal design of experiments for the synthesis of PDIB. The number in brackets indicate their level in the experimental design. Experiment ID 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Tp 95 (2) 105 (4) 105 (4) 100 (3) 90 (1) 100 (3) 105 (4) 90 (1) 110 (5) 90 (1) 110 (5) 95 (2) 100 (3) 95 (2) 110 (5) 110 (5) 105 (4) 90 (1) 110 (5) 100 (3)

Factors and their levels Te tp te 95 (2) 10 (1) 20 (3) 90 (1) 30 (2) 15 (2) 90 (1) 10 (1) 10 (1) 90 (1) 40 (4) 15 (2) 97 (3) 10 (1) 10 (1) 97 (3) 20 (2) 10 (1) 95 (2) 20 (2) 15 (2) 95 (2) 40 (4) 15 (2) 90 (1) 10 (1) 15 (2) 95 (2) 30 (3) 15 (2) 95 (2) 20 (2) 10 (1) 97 (3) 10 (1) 15 (2) 90 (1) 30 (3) 20 (3) 90 (1) 30 (3) 10 (1) 90 (1) 10 (1) 15 (2) 95 (2) 30 (3) 10 (1) 90 (1) 40 (4) 10 (1) 90 (1) 20 (2) 20 (3) 97 (3) 40 (4) 20 (3) 95 (2) 10 (1) 10 (1)

R 1 (2) 2 (3) 1 (2) 0.5 (1) 0.5 (1) 1 (2) 0.5 (1) 1 (2) 2 (3) 1 (2) 1 (2) 1 (2) 1 (2) 0.5 (1) 1 (2) 0.5 (1) 1 (2) 2 (3) 0.5 (1) 2 (3)

2.4. FTIR and 1H-NMR The replacement of hydroxyl groups and formation of ester groups was confirmed via FTIR spectroscopy. Spectra were collected at the resolution of 2 cm-1, and an average of 64 scans in each interferogram on a Varian 660-IR using a PIKE Miracle single reflection attenuated total reflectance with diamond/ZnSe crystal. The 1H-NMR spectra of the material in deuterated methanol were recorded by a Brucker DPX 500 MHz spectrometer, operating at 500 MHz for 1H. The chemical shifts are expressed in δ as parts per million (ppm).


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2.5. Gel Permeation Chromatography (GPC) The molecular weight of the final products was determined through gel permeation chromatography (GPC). All measurements were performed via a UFLC Shimadzu Prominence GPC machine using dimethylacetamide (DMAC) with butylated hydroxytoluene (BHT) (0.05 w/v%) and lithium bromide (LiBr) (0.03 w/v%) at 50 °C and flow rate of 1 ml min-1. 2.6. Rheological properties of PDIB Rheological properties of the synthesised polymers were determined to establish a relationship between viscosity and the reaction parameters such as time and temperature of polycondensation. The dynamic viscosities of synthesised PDIB were measured using an Anton Paar rheometer (Physica MCR 301, Germany). Shear rate sweeps were measured at controlled temperature sets from 20 and 40 °C with the linear shear rate ramping from 0 to 100 s-1 in 2 min. For each shear rate, the average of three measurements was reported as the dynamic viscosity. The frequency sweep was performed by varying the angular frequency from 1 to 100 rad s-1 at 25 and 37 °C to measure the stress and loss moduli of PDIB and their rheological dependency on angular frequency. To further understand the physicochemical properties of PDIB, the effect of temperature on the viscosity of the material was investigated and described through the Arrhenius expression as follow 26 : 𝐸𝑎

𝑘 = 𝐴𝑒

( ― 𝑅𝑇)

(1)



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Where A is the pre-factor (Pa s), k is the apparent viscosity, Ea is the activation energy (kJ mol1),

R is the gas constant (8.314 J mol-1 K-1), and T is the absolute temperature (K). Equation (1)

can be rewritten in a linear form as follows:

ln (𝑘) = ln (𝐴) ―

𝐸𝑎 1 R 𝑇

()

(2)

Therefore, the activation energy and the pre-factor could be estimated from the slope and the intercept of ln (k) versus 1/T, respectively. The viscosity of the PDIB samples measured at 20, 25, 30, 35, 40 °C, and a shear rate of 100 s-1 was used in Equation (2). Adhesiveness of the PDIB samples was measured using the Anton Paar rheometer (Physica MCR 301, Germany) with a flat stainless-steel spindle (25 mm diameter) at 25 °C. PDIB samples (0.05 ml) were placed between the departing parallel plates of the rheometer (500 μm initial gap) and the maximum normal force at which the sample detached from the plates was recorded as the adhesive force. 2.7. In vitro drug release studies (Bradford assay) Bovine serum albumin (BSA)-loaded (100ul solution in water) PDIB samples (0.9 ml) were injected into 10 ml vials, along with 5 ml of release buffer (10 mM PBS pH 7.4, 150 mM NaCl) and stored in an incubator at 37 °C for 60 days. Periodically, 5 μl of the supernatant was taken and replaced with fresh buffer at pre-determined time points. The supernatant sample was mixed with Bradford reagent27 (195 μl) in a 96 well-plate and incubated at room temperature for 20 minutes. The concentration of BSA in all samples was determined using UV-VIS spectroscopy (Biorad 680) at 595nm. All measurements were performed in triplicates and the dilution factor were assumed to be negligible (0.03% per sample).


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Intra-muscular implantation of injectable carriers Female C57BL6 mice aged 8 weeks were purchased from the Animal Resources Centre (Western Australia, Australia) and kept onsite in a Specific Pathogen Free (SPF) animal facility. Animals were allowed access to chow and water ad libitum. Mice were allowed to acclimatize to the facility for 1 week prior to surgery. Ethics for the experiment was approved by the CHW/CMRI Animal Ethics Committee (Protocol number K294). The injectable carriers were prepared by initially dissolving with 20% ethanol (20% v/v for SAIB, and 20% w/w for PDIB) and allowed to dissolve thoroughly to create stock solutions. Immediately prior to surgery, the stock solutions of carrier were mixed with BMP-2 and these were then loaded into 1 mL Luer lock disposable syringes (BD Australia, North Ryde, Australia). Animals were pre-dosed with 0.05 mg/kg buprenorphine 30 minutes prior to implantation for pain relief. Mice were then anaesthetized with inhaled isoflurane gas. The surgical site was sprayed with 70% ethanol, and wiped. Using a 27G needle, 20 µL of the carrier/BMP-2 solutions were injected bilaterally into the quadriceps of the mouse hind limb. Mice were then allowed to recover from anaesthetic under close monitoring. For the preliminary toxicity study, 20 µL of each carrier (SAIB, PDIB) containing 5 µg BMP-2 per injection was delivered a single mouse (n=1 per carrier). Mice were closely monitored for any signs of implant toxicity, and were culled using a CO2 gas chamber after one week. In the bone formation assay, 20 µL SAIB and PDIB containing 5 µg BMP-2 were delivered bilaterally to experimental mice (n=5 mice per carrier). Mice were monitored post operatively, and culled after three weeks using a CO2 gas chamber. In both studies, the sites of interest were harvested after the



cull and preserved in 10% formalin for 4 hours at room temperature, then overnight at 4°C. Samples were subsequently stored in 70% ethanol. Radiographic analysis Bone nodule formation was monitored using digital X-ray (Faxitron X-ray Corp, Illinois, USA), including an end point X-ray at harvest (25 kV, 2× magnification). Samples were next scanned via micro-computed tomography (µCT) using a SkyScan 1174 compact µCT scanner (Kontich, Belgium). Samples were scanned in saline soaked gauze, using a 0.5 mm aluminium filter, 50 kV X-ray tube voltage, and 800 µA tube electric current. Bone pellets were scanned at a pixel resolution of 14.8 µm. The scanned images were reconstructed using NRecon (SkyScan), and analysed with the accompanying software package CTAnalyser (version 1.13.5.0, SkyScan). Statistical methods Statistical analyses were carried out using GraphPad Prism (Version 6; GraphPad Software, CA, USA). For the analysis of bone volume, multiple comparisons were made using a Kruskal Wallis test, followed by post-hoc Mann Whitney U testing. Non-parametric tests were selected as bone volume in muscle pouch studies do not typically follow a normal distribution. Statistical significance was set at α

Optimized Synthesis of Poly(deoxyribose) Isobutyrate, a Viscous

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