Generational biodegradable and regenerative polyphosphazene polymers and their blends with poly (lactic-co-glycolic acid)

Journal Pre-proof Generational Biodegradable and Regenerative Polyphosphazene Polymers and their Blends with Poly (lactic-co-glycolic acid) Kenneth S. Ogueri, Harry R. Allcock, Cato T. Laurencin

PII:

S0079-6700(19)30107-8

DOI:

https://doi.org/10.1016/j.progpolymsci.2019.101146

Article Number:

101146

Reference:

JPPS 101146

To appear in:

Progress in Polymer Science

Received Date:

14 March 2019

Revised Date:

19 July 2019

Accepted Date:

2 August 2019

Please cite this article as: Ogueri KS, Allcock HR, Laurencin CT, Generational Biodegradable and Regenerative Polyphosphazene Polymers and their Blends with Poly (lactic-co-glycolic acid), Progress in Polymer Science (2019), doi: https://doi.org/10.1016/j.progpolymsci.2019.101146

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Generational Biodegradable and Regenerative Polyphosphazene Polymers and their Blends with Poly (lactic-co-glycolic acid)

Kenneth S. Ogueria, b, Harry R. Allcockc, d, Cato T. Laurencina, b, e, f, g*.

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Graphical abstract

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*Corresponding Author: [email protected]

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a. Department of Materials Science and Engineering, University of Connecticut, Storrs, CT 06269, USA b. Connecticut Convergence Institute for Translation in Regenerative Engineering, University of Connecticut Health Center, Farmington, CT 06030, USA c. Department of Chemistry, The Pennsylvania State University, University Park, PA 16802, USA d. Department of Materials Science and Engineering, The Pennsylvania State University, University Park, PA 16802, USA e. Department of Orthopaedic Surgery, University of Connecticut Health Center, Farmington, CT 06030, USA f. Department of Biomedical Engineering, University of Connecticut, Storrs, CT 06269, USA g. Department of Chemical and Biomolecular Engineering, University of Connecticut, Storrs, CT 06269, USA

Abstract New fields such as regenerative engineering have driven the design of advanced biomaterials with a wide range of properties. Regenerative engineering is a multidisciplinary approach that integrates the fields of advanced materials science and engineering, stem cell science, physics, developmental biology, and clinical translation for the regeneration of complex tissues. The complexity and demands of this innovative approach have motivated the synthesis of new polymeric materials that can be customized to meet application-specific needs. Polyphosphazene polymers represent this 1

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fundamental change and are gaining renewed interest as biomaterials due to their outstanding synthetic flexibility, neutral bioactivity (buffering degradation products), and tunable properties across the range. Polyphosphazenes are a unique class of polymers composed of an inorganic backbone with alternating phosphorus and nitrogen atoms. Each phosphorus atom bears two substituents, with a wide variety of side groups available for property optimization. Polyphosphazenes have been investigated as potential biomaterials for regenerative engineering. Polyphosphazenes for use in regenerative applications have evolved as a class to include different generations of degradable polymers. The first generation of polyphosphazenes for tissue regeneration entailed the use of hydrolytically active side groups such as imidazole, lactate, glycolate, glucosyl, or glyceryl groups. These side groups were selected based on their ability to sensitize the polymer backbone to hydrolysis, which allowed them to break down into non-toxic small molecules that could be metabolized or excreted. The second generation of degradable polyphosphazenes developed consisted of polymers with amino acid ester side groups. When blended with poly (lactic acid-co-glycolic acid) (PLGA), the feasibility of neutralizing acidic degradation products of PLGA was demonstrated. The blends formed were mostly partially miscible. The desire to improve miscibility led to the design of the third generation of degradable polyphosphazenes by incorporating dipeptide side groups which impart significant hydrogen bonding capability to the polymer for the formation of completely miscible polyphosphazenePLGA blends. Blend system of the dipeptide-based polyphosphazene and PLGA exhibit a unique degradation behavior that allows the formation of interconnected porous structures upon degradation. These inherent pore-forming properties have distinguished degradable polyphosphazenes as a potentially important class of biomaterials for further study. The design considerations and strategies for the different generations of degradable polyphosphazenes and future directions are discussed.

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Keywords biomaterials, biodegradable polyphosphazene, regenerative engineering, biocompatibility, poly (lactic-co-glycolic acid)

Nomenclature ALP

Differential Scanning Calorimetry

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DSC

Alkaline Phosphatase

FTIR

Fourier Transform Infrared

HCCTP

Hexachlorocyclotriphosphazene

PDCP

Poly (dichlorophosphazene)

PLGA

Poly(lactic-co-glycolic acid)

PNGEGPhPh Poly[(glycineethylglycinato)50(phenylphenoxy)50phosphazene] SDH

Succinic Dehydrogenase 2

1. Introduction The development of synthetic biodegradable polymers for a variety of biomedical applications such as regenerative engineering scaffolds, therapeutic drug delivering systems, and sutures have witnessed an increasing interest.[1-3] The properties of materials need to be tailored to meet specific requirements for various disease states. [4-8] Polymeric biomaterials can be an essential

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component for developing 3D matrices for tissue regeneration. [4, 5, 8, 9] The suitability of

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polymers for use in matrices is based on their compatibility with living cells and the ability to decompose when exposed to aqueous media.[8, 10] Commonly used biodegradable polymers

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include polyesters, polyorthoesters, polyanhydrides, poly (amino acids), and polyphosphazenes [8,

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11-13]. Even though polyphosphazenes are a relatively new and emerging technology among the list, they offer an exceptionally attractive platform for the design of a new class of biodegradable

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polymers that exhibit neutral bioactivity and a broad spectrum of material properties (such as degradation rates and mechanical properties). [8, 13-17] More neutral degradation product pH can

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be highly desirable in tissue regeneration as few degradable polymers exhibit this feature. A few specialized polyphosphazenes, mainly, those with hydrolytically active side groups have been designed and investigated as matrices for biomedical applications such as bone tissue regeneration. [7, 11, 18-20]

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Polyphosphazenes are a class of inorganic-organic hybrid polymers with a backbone of alternating phosphorus and nitrogen atoms and with two organic sides directly bonded to the phosphorous atom. [21, 22] They are widely produced using a two-step reaction process with a commercially available starting material, hexachlorocyclotriphosphazene (HCCTP). [22, 23] The first step involves the thermal ring-opening polymerization of the HCCTP to obtain a highly reactive

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macromolecular intermediate, poly (dichlorophosphazene) (PDCP). [22-24] The second step, termed the macromolecular nucleophilic substitution is subsequently carried out by the substitution of

chlorine

atoms

of

PDCP

with

alkoxides,

aryloxides,

or

amines

to

yield

poly(organo)phosphazenes.[25] Depending on the chosen side groups and compositions, Poly(organo)phosphazenes constitute a variety of materials with a wide range of properties that

made them the subject of keen interest in regenerative engineering.[18]

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can find their place in numerous applications. The synthetic flexibility and property tunability have

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In the quest for appropriate biomaterials for tissue regeneration, various biocompatible and

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hydrolytically sensitive organic side groups have been employed in the synthesis of polyphosphazene-based biomaterials as regenerative matrices. [18, 19, 26-35] This pressing need

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has led to the evolution of different generations of degradable polyphosphazenes as potential biomaterials for matrix-based regenerative engineering. The pioneering attempts to design

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degradable polyphosphazene for tissue regeneration led to the development of the first generation which includes the use of side groups such as imidazole, lactate, glycolate, glucosyl, or glyceryl.

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[18, 19, 36, 37] These groups were capable of allowing the hydrolytic breakdown of polyphosphazene backbone into a pH-buffered system of phosphate and ammonia; however, they recorded moderate cell growth and proliferation.[36]

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The second generation of degradable polyphosphazene was designed using amino acid esters. [36, 38-40] The hydrolytic degradation of poly [(amino acid ester) phosphazenes] resulted in biologically benign products which included amino acid groups, alcohol from the ester, phosphate, and ammonia. [11, 18, 40] In a blend with poly (lactic acid-co-glycolic acid) PLGA, degradation products exhibited a near neutral pH; however, the blend miscibility was partial. [31, 32] The third generation was developed to surmount the miscibility problem by using organic side groups with 4

high hydrogen bonding capabilities. Peptide esters were found to be ideal moieties, as they provide more hydrogen bonding sites for miscibility as compared to amino acid esters. [27, 30, 41, 42] Similar to amino acid ester based polyphosphazenes, the third generation showed robust cell growth and proliferation.[30] A unique degradation mechanism was seen for dipeptide-based polyphosphazene-PLGA blends as they possessed inherent pore-forming properties. [27, 33] A

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study by Deng et al. demonstrated the ability of these blends to form an assemblage of microspheres with interconnected porous structures upon degradation.[33]

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This review gives an overview of the different generations of degradable polyphosphazenes that

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2. Regenerative Engineering Concepts

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polyphosphazene polymers for tissue regeneration.

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have been investigated as matrix materials and as well as ongoing work and the future outlook for

The success of matrix-based regenerative engineering is dependent upon the biomimetic scaffold

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that provides a structural template for tissue regeneration.[4] Biodegradable polymers are an indispensable component of this approach as it can be used to create scaffolds for tissue development.[27, 33, 43] Regenerative engineering presents a flexible and reliable toolbox that enables the regeneration of complex tissue and organs.[4, 44] This toolbox combines expertise in

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advanced materials science & engineering, stem cell science, physics, developmental biology, and clinical translation to guarantee positive clinical outcomes.[4] Advanced material science & engineering ensures the design of appropriate biomaterial for specific regenerative requirements. The biomaterials ideally are expected to degrade at a rate that matches the process of regeneration, have resorbable degradation products, and provide adequate initial mechanical support and

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structural integrity, while modulating the cellular activities by presenting chemical and biochemical cues with precise control.[33] The inducement of tissue regeneration and bone healing with signaling and bioactive molecules is essential to regenerative engineering as it constitutes the morphogenesis and developmental biology.[4] Morphogenesis and dynamics of tissue development depend on the spatial and

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temporal control of the cells’ mechanics. Thus, biomechanical stimulations can coordinate cellular activities towards the development of tissues.[5] The mechanics of cells requires an understanding

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of physics principles of mechanics of structures and the integration of force and stress within the

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substrate during the sequential morphogenic steps of tissue regeneration. Stem cell technology is vital to regenerative engineering approach as it combines with extracellular matrix scaffolds and

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inductive signals to produce tissue-engineered constructs.[45] Stem cells have high clinical prospects due to their ability to differentiate into various lineage tissues with multiple cell types.

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Bio-instructive scaffolds support the stem cell differentiation and modulate their response to bioactive stimuli. Biomaterials with an appropriate combination of physical, mechanical, and

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chemical properties could facilitate desirable cellular responses and promote mineralized matrix synthesis.[45] Clinical translation creates an avenue to harness and maximize the symbiosis of the expertise mentioned above and translate them into the much needed clinical applications. It utilizes

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the outcome of the research done in the laboratory to develop innovative ways to treat patients. [4, 5]

This exciting new field of regenerative engineering will require the development of new advanced biomaterials with unique properties across the range that can satisfy a variety of tissue regenerative needs. Polyphosphazenes can have a crucial role to play in this new approach of tissue regeneration

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as they offer a versatile platform for well-defined tunability in terms of degradability, biocompatibility, and mechanical properties. [11, 18, 21, 25, 26, 46] 3. Macromolecular Nucleophilic Substitution Macromolecular substitution provides a platform on which different organic groups can be used to substitute chlorine atoms of the PDCP intermediates, resulting in a variety of poly (organo

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phosphazenes) with a spectrum of properties.[21, 22, 25] This offers an advantage over other classical organic polymers or silicones. The labile nature of the P–Cl bond is the driving force for

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this substitution reaction and the ease with which it occurs depends on factors such as reaction

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temperature and time, type of nucleophiles (nucleophilicity, and steric hindrance), type of solvent, and solubility of byproducts.[11, 18, 21, 22, 25, 26] The chemistry of macromolecular substitution

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as shown in figure 2 allows the synthesis of not only single-substituent polyphosphazenes with

lP

one type of side groups but mixed-substituent polyphosphazenes (copolymers) with two or more different side groups.[8, 11, 23] The halogen substitution can be achieved simultaneously or sequentially and the arrangement of the side groups linked to the backbone of mixed-substituent

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polyphosphazenes can be specified with reaction conditions.[11, 21, 22, 47, 48] The P-N backbone and the nature of each side group or combination of side groups determine the characteristics of polyphosphazenes. [22, 25] The backbones of these polymers possess unique

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features as they contain inorganic elements. [11, 18, 21, 22, 24] The use of organic side groups that are hydrophilic in nature (such as amino acid esters, imidazolyl, and glucosyl, glyceryl, and glycolate or lactate ester) results in poly(organo)phosphazenes that undergo degradation. The presence of hydrolytically active side groups would induce hydrolysis within the polyphosphazene backbone and thereby causing a breakdown to ammonia, phosphate and corresponding side groups. [18, 24, 49] On the other hand, hydrophobic side groups would protect the skeleton from 7

hydrolysis. [18] This is of interest in designing bioerodible polymers for regenerative engineering and provides scope and perspective for the optimization of degradation and mechanical properties. The type of side groups selected can affect the torsional mobility of the backbone and hence the glass temperature of the resulting poly (organo) phosphazene. For example, a bulky side group (such as aryloxy groups) will hinder or restrict the rotation of the backbone, resulting in polymers with high glass transition temperatures. In contrast, small groups (such as alkyl and fluoroalkoxy

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side groups) would cause less or no restriction on the mobility of the backbone, thereby amounting

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to a flexible polymer with a low glass transition temperature. Therefore, the properties are highly

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dependent on the side-substituents and their ratios. [8, 11, 18, 22, 49, 50]

The interaction of the polymer molecules with each other, other polymers (in the case of a blend

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system), and the environment can depend on the type of side groups utilized. Biocompatible side groups are desirable in the design of polyphosphazenes intended for use in tissue regeneration.

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[18, 21, 26] This allows the favorable interaction of the polymers with the surrounding cells and tissue in the microenvironment and the production of non-toxic degradation products (the

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corresponding side groups, ammonia and phosphates). [11, 49, 50] Another important aspect of the macromolecular substitution is to ensure the complete replacement of the chlorine atoms of the PDCP with the intended organic nucleophiles.[11, 18] Hydrolytic

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instability and uncontrollable crosslinking/or degradation tendencies can result from unreacted P– Cl in the polymer chain and can be detrimental to cells and tissues as P–Cl may react with surrounding water in the tissue microenvironment to form P–OH units and hydrochloric acids.[18] The hydrochloric acid produced is usually incompatible with living cells and tissues.[11, 18] Monitoring the disappearance of peaks characteristic of P-Cl units with

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31

P NMR, using a

stoichiometric excess of the organic nucleophile, and allowing sufficient reaction time, are strategies that can be used to ensure the complete substitution of the chlorine atoms. [11, 22, 48] In addition to the factors and strategies mentioned above that can affect the substitution of chlorine atoms, small molecule model reactions

can be utilized to examine the feasibility of the

macromolecular substitution of each selected organic nucleophile under consideration.. This

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feasibility study can be done by reacting HCCTP with each of the intended organic side groups

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and monitoring the facile interactions of the two compounds with 31P NMR (see figure 3).[48] The type of linkage to the polyphosphazene backbone can affect degradation rates of the polymer.

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[11, 18, 21, 22, 24] In near-neutral media, polyphosphazene polymers with side groups linked through oxygen are more resistant to hydrolysis than polymers with side groups linked through

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nitrogen of a primary or secondary amine. [18, 22, 26] This behavior is attributable to the relative

lP

stability of P–O–R bonds and hence aryloxy and alkoxy units are employed as co-substituents in

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macromolecular substitution to modulate degradation rates. [8, 18, 51]

4. First Generation Degradable Polyphosphazenes The motivation to explore polyphosphazenes in the field of biomedicine stemmed from the following facts: 1. Polymers with a wide range of properties can be synthesized with the same

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starting material (HCCTP to PDCP) and by changing the side chain substituents 2. Specific side chain substituents, especially hydrolytically sensitive ones can impose hydrolytic instability to the backbone of the polymer 3. The products of the degradation were found to be benign and consist of ammonia, phosphate, and corresponding side groups.[40, 52]

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The first generation of degradable polyphosphazenes designed for use in tissue regeneration consists of polyphosphazene polymers with side groups such as imidazole.[36] The selection of this particular side group was based on its biocompatibility and its ability to confer hydrolytic instability to the backbone. The resulting degradation products would be non-toxic and neutral since Imidazoles are amphoteric. [11, 18, 53] Laurencin et al.[36] in collaboration with the Allcock research group pioneered the investigation of polyphosphazene-based polymers as potential

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polymeric support for cells in tissue regeneration. Poly [(imidazolyl) (methylphenoxy)

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phosphazenes] was synthesized and used in their study, and their ability to sustain osteoblast like MC3T3-E1 cell growth was examined after 3 and 7 days in culture. The MC3T3-E1 cells exhibited

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spindle-like morphology and moderate adhesion on the samples after 2 days of culture. Overall,

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the Poly [(imidazolyl) (methylphenoxy) phosphazenes] samples when seeded with MC3T3-E1 cells showed enhanced cell attachment and proliferation after 7 days.[36] Alkaline phosphatase

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(ALP) activity for osteoblast phenotype expression was significantly enhanced on the polymers as compared to PLGA. However, an increase in the content of imidazolyl side groups of the

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polyphosphazene resulted in a decrease in cell attachment and growth. The presence of the imidazolyl groups linked to the backbone caused the polymers to degrade and the degradation rate was found to increase as the content of the imidazolyl side group in the polyphosphazene polymer was increased.[36] The incubation of MC3T3-E1 cells with the degradation products of the

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degradable imidazolyl-substituted polyphosphazene had no negative impact on cell growth except for cells incubated with the degradation products of polymers with high imidazolyl content (54%). The results of the study by Laurencin et al. indicated that changing the concentration of imidazolyl groups in the polymer may influence cell growth as cell attachment and proliferation decreased with increasing imidazolyl content. While at the same time, polymer degradation rates increased

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with increasing content of imidazolyl side groups. These characteristics appeared to be modulated by the nature of the side chain and its percentage substitution. Results obtained for poly [(imidazolyl) (methylphenoxy) phosphazenes] samples were compared to that of the amino acid ester-substituted polyphosphazenes.[36] Substitution with amino acid esters constitute the second generation of degradable polyphosphazenes, and this will be addressed in the next section.

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5. Second Generation Degradable Polyphosphazenes The linkage of amino acid esters to the polyphosphazene backbone via the amino terminus imparts

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an essential set of properties to polyphosphazenes. Similar to the imidazolyl-substituted

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polyphosphazenes, these polymers are hydrolytically sensitive and degrade to phosphate, ammonium ion, and the corresponding side group, which in this case are amino acid, and

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ethanol.[54] The rates at which these amino acid ester-substituted polymers degrade depend on the specific amino acid employed and ester unit present.[26, 42, 54] This is often controlled by the

lP

hydrophobicity and steric hindrance of the substituent at the α-carbon of the amino acid and the ester unit. For instance, amino acid esters with a bulky substituent at the α-carbon (such as in

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phenylalanine) with a long alkyl chain of the ester segment are expected to result in amino acid ester-substituted polyphosphazenes with slow degradation. On the contrary, glycine methyl ester with just a hydrogen atom on the α-carbon and short ester segment is expected to exhibit

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accelerated degradation.[8] Another critical aspect in the choice of amino acids is to ensure the mono-functionality of the intended organic nucleophiles during the halogen replacement. Thus, amino acid esters are preferred to just the amino acid to prevent crosslinking of the system during macromolecular substitution as the carboxylic acid moiety is esterified, and the primary amine is then utilized only for attachment to the phosphorus atoms.[18] An early study by Laurencin on the feasibility of using polyphosphazenes as matrices for tissue regeneration drew a comparison 11

between amino acid ester-substituted polyphosphazenes and imidazolyl-substituted ones mentioned above. The suitability of poly [ethyl glycinato) (methylphenoxy) phosphazenes for bone repair was studied and compared to poly [(imidazolyl) (methylphenoxy) phosphazenes. Both polymers supported the growth of MC3T3-E1 cells but unlike the poly [(imidazolyl) (methylphenoxy) phosphazenes, an increase in the content of the ethyl glycinato group favored increased cell attachment and growth. Also, hydrophilicity and degradation rates of the poly [ethyl

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glycinato) (methylphenoxy) phosphazenes were increased with increasing content of ethyl

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glycinato moiety. Since this early study by Laurencin et al., numerous studies have demonstrated the biocompatibility of amino acid ester-substituted polyphosphazenes.[54] In one of such studies, et

al.

[55]demonstrated

the

biocompatibility of

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Gumusderelioglu

poly[bis(ethyl

4-

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aminobutyro)phosphazene] using cytotoxicity tests in which the undesired effects of the residuals on the living tissues are determined. For these experiments, the activity level of succinic

lP

dehydrogenase (SDH) of Swiss 3T3 and HepG2 cell lines in the polymer extracts was analyzed by MTT assay method as SDH acts as a good indicator of cytotoxicity because of its significant

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involvement in cellular metabolism. The results revealed high SDH activities for all time points, suggesting that the extracts of the amino acid-based polyphosphazene products did not negatively affect the viability of either cell. In another study, Carampin and coworkers investigated the cytocompatibility of cosubstituted poly (amino acid ester) phosphazene electrospun nanofibers rat

endothelial

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towards

cells.[56]

They subjected

poly[(ethyl

phenylalanato)1.4(ethyl

glycinato)0.6phosphazene to adhesion and proliferation assays and found that there was an improvement in the cell adhesion and proliferation as compared to fibronectin coated polystyrene tissue culture plate. Amino acid ester-substituted polyphosphazenes have been employed in the design of blend systems with polyesters such as PLGA and polylactic acid (PLA).[57] PLGA and

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PLA are the most extensively investigated biodegradable polymers for bone tissue regeneration due to their well-established biocompatibility and commercial availability, however, the issue of acidic accumulation of their degradation products (glycolic and lactic acids) due to bulk erosion, have hampered its clinical effectiveness as the acids can in some cases cause inflammatory responses, foreign body reactions, and unexpected structure failure.[8, 58] Thus, the quest for polymer systems with neutral bioactivity and degradation products fueled the development of

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amino acid ester based polyphosphazene-polyester blends. Deng et al.[32] demonstrated the ability

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of ethyl glycinato substituted polyphosphazenes to neutralize the acidic degradation products and control the degradation rate of PLGA by blending. The blends exhibited excellent cell proliferation

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and ALP activity as compared to PLGA (figure 4). However, the blends with ethyl glycinato-

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substituted polyphosphazene composition above 25% were partially miscible as scanning electron microscopy (SEM) indicated phase separations and differential scanning calorimetry (DSC)

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depicted two glass transition temperatures for each of the blends. Immiscibility of the polymer components affects the mechanical properties of the resulting blends. Another study by Nair et al.

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[59] showed that degradation products of poly [bis(ethyl alanato) phosphazene] (PNEA) could effectively neutralize the acidic degradation products of PLGA. The blend films showed significantly higher cell numbers on the surface compared to PNEA and PLGA films. However, based on the DSC, SEM, and Fourier-transform infrared spectroscopy (FTIR), PNEA was not

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wholly miscible with PLGA. Moreover, many other studies have proven the ability of amino acid ester substituted polyphosphazenes to neutralize the acidic degradation products of polyesters. [10, 19, 20, 25, 41, 52, 54, 60-67] The third generation polyphosphazenes were born out of the quest to address the miscibility issue of amino acid ester substituted polyphosphazenes when blended with PLGA. [36, 41, 42, 68]

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6. Third Generation Degradable Polyphosphazenes Miscibility is a critical factor in achieving uniform polymeric matrices with predictable properties and efforts have been made in addressing the issue of miscibility in polyphosphazene-polyester blend systems. In one

such study, Weikel et al.[69] designed a block copolymer of

polyphosphazene-polylactide segments whose purpose was to act as compatibilizers in creating

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miscible blends of amino acid ester-ester substituted polyphosphazene and PLGA. A miscible blend could be attained by incorporating small quantities (approximately 5 – 7.5 % wt.) of the

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block copolymer. However, it is a rigorous synthetic challenge to make polylactidepolyphosphazene block copolymers because polylactides and polyphosphazenes have different

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polymerization chemistries. In the synthesis of the block copolymer, the polyester segment is first

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linked to the backbone of the PDCP, and then the chlorine atoms in the PDCP are subsequently substituted with the amino acid esters. Unexpected decomposition of the phosphazene blocks or

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sudden cleavage of the phosphazene-polyester linkage may occur as a result of the generation of HCl byproducts during the replacement of chlorine atoms. [21, 22, 48]

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In order to circumvent the use of this more complicated procedure, the concept and feasibility of blending PLGA with dipeptide ester-substituted polyphosphazenes was first proposed by Laurencin’s research group. [30, 41, 60] Dipeptide esters (such as glycylglycine ethyl ester)

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possess a higher number of hydrogen bonding sites than amino acid esters and hence possess a higher capability of forming intermolecular hydrogen bonds with carbonyl groups of the PLGA molecules when used as side groups for polyphosphazenes (figure 5). These intermolecular interactions could foster micro-scale blend miscibility between the two individual polymers.[68] Studies by Krogman et al.[68] and Deng et al.[30] demonstrated that increased hydrogen bonding between dipeptide ester-substituted polyphosphazenes and polyesters leads to completely miscible 14

blends and the effective neutralization of the acidic degradation products of PLGA by the polyphosphazene hydrolysis products (see figure 6, 7, & 8a). As shown in Figure 6a, the exhibition of single glass transition temperature by the polyphosphazene-PLAGA blends indicated complete miscibility and the glass transition temperatures of the miscible blend were found to be the intermediate between those of the two original polymers[30]. The complete miscibility achieved through intermolecular hydrogen bonding can enhance mechanical properties if adequately

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optimized. Based on Fourier transform infrared (FTIR) spectroscopy, the IR band for the

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intermolecular hydrogen bonds between the secondary amine of dipeptide ester-substituted polyphosphazenes, and carbonyl groups of the polyesters occurs around 1677 cm-1 (Figure 6b).[30]

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Another study by Krogman et al.[70] demonstrated the possibility of attaining a miscible blend of

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polyphosphazene and PLGA by utilizing other side groups such as Tris (hydroxymethyl) amino methane (THAM). THAM was found to have a sufficient amount of hydrogen bonding sites

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through its hydroxyl groups.

Because compatibility to cells is one of the critical criteria a polymer must meet in order to be

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suitable for regenerative engineering, dipeptide substituted polyphosphazenes, and their blends with polyesters have been proven to be biocompatible and to support cell growth. [13, 20, 26, 28, 30] In a rat model, two subcutaneously implanted blends of poly [(glycineethylglycinato)1(phenyl

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phenoxy)1phosphazene] (PNGEGPhPh) and PLGA were monitored for biocompatibility through an inflammatory response.[30] After 12 week implantation, the blends showed enhanced biocompatibility as compared to PLGA. There was no evidence of physical impairment observed on the blends during the 12-week post-implantation period; however, the blend implants were surrounded by a relatively thinner fibrous capsule than PLGA. As indicated in Figure 7 a-f, the blends showed thinner fibrous capsules than PLGA. There was a decrease in the inflammatory 15

responses with time for the blends, whereas inflammatory response increased for PLGA. The thickness of the inflammatory zone for the blends was thinner than that of the PLGA sites after 2 weeks (Figure 7a-c). A further decrease was observed in the thickness of the inflammatory zones for blends’ implantation sites after 4 weeks while the inflammatory zone of the PLGA site got more significant in size at the same time point (Figure 7d-f). This drastic increase in the PLGA’s inflammatory response is as a result of its bulk degradation to lactic acid and glycolic acids[30].

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Not only were the dipeptide ester-substituted polyphosphazene-PLGA blends found to exhibit

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excellent biocompatibility and exceptional buffering effect (figure 7a-f), but a unique erosion mechanism was establish in the blended matrices ( figures 8c & 8d).[33] Upon degradation, the

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blends change from a coherent solid into 3-D porous structures composed of spheres and pores of

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up to 10-100μm (see figure 8c).[33] This type of erosion is quite distinctive, and its mechanistic pathways have been proposed by Deng et al. to happen in three sequential stages. The first stage

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involves the breakdown of the intermolecular hydrogen bonding between the dipeptide of the polyphosphazene and the carbonyl groups of the PLGA. Intra-molecular hydrogen bonds between

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polyphosphazene chains are formed in the second stage resulting in the rearrangement of the polyphosphazenes into spheres. The final stage entails the breakdown of the intra-molecular hydrogen bonding interactions of the polyphosphazene molecules which results in the final degradation of the blend system. FTIR has confirmed the sequence of these three erosion stages

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in this study.[33] The in-situ pore-forming ability of the dipeptide ester-substituted polyphosphazene-PLGA blend is exciting in that it presents the prospects of creating a matrix that obviates the need to prefabricate porous 3-D structures. Often, 3-D porous structures are characterized by poor initial mechanical properties unsuitable for the regeneration of bone tissues (see figure 8c & 8d). The utilization of the dipeptide esters as a substituent for polyphosphazenes

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presents important possibilities for the effective optimization of polyphosphazene-polyester blends through the unique dipeptide chemistry with its extensive hydrogen bonding sites, and the in-situ pore-forming ability of this class of blend. There is now a greater possibility of using a full range of tools such as the side group chemistry and blend compositional changes to tailor material

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properties for specific needs and applications in areas such as regenerative engineering.

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7. Ongoing work and future outlook on Polyphosphazenes for Regenerative Engineering Current work is focused on the development of high strength polyphosphazenes by utilizing

practical regenerative applications.

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dipeptide chemistry to fine-tune the properties of dipeptide co-substituted polyphosphazenes for The selection of dipeptide-based side groups and co-

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substitution with moieties that possess the ability to enhance hydrogen bonding to PLGA further

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may impart enhanced mechanical features to the overall blend system. In other words, the synthetic flexibility of dipeptide cosubstituted polyphosphazenes will allow the design of specific side group chemistry that is expected to enable strong hydrogen bonding interactions as well as mechanical

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integrity. Meanwhile, the hydrogen bonding interactions found in dipeptide molecules are similar to nature’s own chemistry as hydrogen bonding significantly contribute to the coiling of proteins and the formation of unique and distinct conformations in biological systems.

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The use of dipeptide would not only impact the mechanical properties of the blend but also will allow for the direct control of the rate and levels of pore formation. Reaction conditions and kinetics offer a platform for understanding the mechanistic effects of polyphosphazene synthesis on the overall degradation patterns and erosion mechanisms of the blend. These conditions and kinetics, which include reaction time, temperature, and sequence of side group substitution, will influence the degradation profile of the blend. Therefore, an ideal polyphosphazene for blending 17

with PLGA could be obtained by microstructural and synthetic manipulations, and the resultant blend matrices through the adjustment of their component compositions could demonstrate superior mechanical performance while optimizing resorptive and biocompatibility properties. 8. Conclusions The rise of regenerative engineering has stimulated a paradigm shift in the design and development

of

of new polymeric materials, whose properties can be personalized to meet application-specific

ro

needs and requirements. Polyphosphazenes provide a springboard for this new development due to its design flexibility and property tunability. The need for continuous improvement in the area

-p

of biomaterial and scaffold design has led to the development of different generations of

re

degradable polyphosphazenes which are summarized as follow.

1. The first generation of polyphosphazenes for tissue regeneration required the incorporation

lP

of hydrolytically active side groups such as imidazole groups. The polymer was degradable, but it witnessed a decrease in the cell number as the imidazolyl content was

ur na

increased.

2. The second generation of polyphosphazenes developed for regenerative applications consisted of polymers with amino acid ester side groups. When blended with PLGA, it showed the feasibility of neutralizing the acidic degradation products of PLGA but formed

Jo

mostly partially miscible blends.

3. The third generation degradable polyphosphazenes was designed by incorporating dipeptide side groups which impart significant hydrogen bonding capability on the polymer for the formation of completely miscible polyphosphazene-PLGA blends. The blends

18

exhibited unique degradation behaviors by which the blends turn from films into an assemblage of microspheres with interconnected porous structures upon degradation. Future work on the development of polyphosphazenes will focus on the control of intramolecular and intermolecular hydrogen bonding capability to create polymer blends with enhanced mechanical properties and controlled resorptive capabilities.

of

Competing interest

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The authors declare no competing financial interest.

-p

Acknowledgments

Support from NIH DP1 AR068147 and the Raymond and Beverly Sackler Center for Biomedical,

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ur na

lP

re

Biological, Physical and Engineering Sciences, are gratefully acknowledged.

19

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3rd Generation Dipeptide containing Polyphosphazenes 2nd Generation Amino acid ester containing Polyphosphazenes 1st Generation Imidazole containing polyphosphazenes P

N

CH3 O HN

O N H

P

CH3

P

O

N

N O

HN CH3

of

O

N

CH3

ro

N

R

R

N

-p

P

R=Organic groups

re

N

The different generations of degradable polyphosphazenes obtained from poly (organo) phosphazene. Cl OR NaOR P N P -NaCl n OR Cl n H2NR

lP

Fig. 1.

ur na

-HCl

NHR N

n

NaOR

-NaCl

P NHR

H2NR

Jo

-HCl

OR N

P n NHR

27

Fig. 2. Mechanism of nucleophilic macromolecular substitution depicting single-substituent and mixed-substituent polyphosphazenes. CH3 O

O (a)

H3C

Cl

P

P

NH

N P

THF, 60 C O

R

N

O

CH3

N H

HN

O

N H HN

O

R

P

P

N

N

NH

O

R

P

Hexachlorocyclotriphosphazene

R NH

O H3C

HN

O

O

O

NH

HN

N

R

Glycylglycine Ethyl Ester After treatment with Triethylamine

Cl

O

R

o

O

H2N

Cl

Cl

Cl

O

Cl

N

NH

O N H

O

CH3

O

O

R

of

HN

O

O

O

(b)

P

Cl

Cl

NaO

N P

Sodium Phenylphenoxide (from Phenylphenoxide and Sodium Hydride)

Cl

lP

Hexachlorocyclotriphosphazene

O

N

P

P

N

N

O

O

P O

ur na

Small molecule model reactions demonstrating the feasibility of selected nucleophilic organic substituents. (a) Glycylglycine ethyl ester reacting with HCCTP (b) Phenylphenol reacting with HCCTP.

Jo

Fig. 3.

O

O

N

Cl

THF, 60oC

-p

Cl

N P

re

Cl

CH3

ro

H3C

28

COOEt HC

R

Glycine + Ethanol Water

R

HC

Hydrolysis

NH N

COOEt

2,5-Diketopiperazine + Ethanol

NH2

Water

P n NH

R

CH

OH

COOEt

N

O

P

P

N n

n

NH

of

R

NH

CH

R

COOEt

CH

ro

COOEt

-p

Chain Opening

PH

Hydrolysis

lP

re

Ammonia + Phosphate

O

NH R

CH COOEt

Jo

ur na

Fig. 4. Degradation mechanism of amino acid ester-substituted polyphosphazenes. The degradation products are ammonia and phosphate which constitute a natural buffer.

29

a

C2H5 O C

O

C2H5

C

O

O

CH2 N O

O H

N O

CH2 H

O

CH2

C

N

C2H5

O CH2

H

N

C O

CH2 N

H

C2H5

C

H

O

CH2

CH2

N

C

O

CH2 N

C

O

H

C

N

C O

H3C

CH2 H

O

O

H

O

C

O

CH2

CH2

N O

O

C

C O

C H3C

CH2

H

O

CH C

ro

N

Intra-molecular hydrogen bonds between peptide molecules

H

CH

of

O

b

C2H5

-p

Intermolecular hydrogen bonds between the peptides and the carbonyl groups of the PLAGA

Jo

ur na

lP

re

Fig. 5. Chemical structures showing the high hydrogen bonding sites for glycylglycine ethyl ester substituent. a) Intra-molecular hydrogen bonding within the peptide molecules b) Inter-molecular hydrogen bonding between the peptide of polyphosphazene and carbonyl oxygen of PLGA.

30

lP

re

-p

ro

of

Fig. 6. Characterizations of miscibility for the blends of poly[(glycineethylglycinato)50(phenylphenoxy)50phosphazene](PNGEGPhPh) and PLGA. a) DSC curves for the blends and individual polymers and the blends exhibit single glass transition temperatures indicating miscibility b) FTIR spectra for the individual polymers and their blends. The peaks at 1677 cm-1 for the blend matrices correspond to the intermolecular hydrogen bonding between the peptide of the polyphosphazene and carbonyl oxygen of the PLGA [30]. Copyright 2010. Reproduced with permission from Elsevier Science Ltd.

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ur na

Fig. 7. Histological representation of the H&E stained sections of PLGA and the blend matrices, indicating the inflammatory response during the post-implantation period. PLGA recorded higher inflammatory responses after 2 and 4 weeks due to the acidic degradation products, whereas the blend matrices showed minimal inflammatory responses as a result of the near-neutral pH of their degradation products [30]. Copyright 2010. Reproduced with permission from Elsevier Ltd.

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of ro -p re

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ur na

lP

Fig. 8. Peptide ester substituted polyphosphazene-PLGA blend a) SEM image of nanofibers composed of miscible blend indicating no phase separation b) Fluorescence image of a live/dead assay of primary osteoblast seeded on the miscible blend after 48 hours. Green color signifies that the cells are living and no evidence of red color which symbolizes dead cells c) Surface morphology of the blend showing the in situ pore forming ability after 12 weeks of implantation d) SEM image of the cross-section of the polyphosphazene spheres indicating smaller pores on them after 10 weeks of implantation. The porosity on the polymer spheres will provide extra surface area and room for more cell-polymer interactions [30, 33]. Copyrights 2010. Adopted with permissions from Elsevier Ltd and John Wiley and Sons Inc.

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