In Vivo Biological Evaluation of High Molecular Weight Multifunctional

Oct 17, 2016 - In Vivo Biological Evaluation of High Molecular Weight Multifunctional Acid-Degradable Polymeric Drug Carriers with Structurally Differ...
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In vivo Biological Evaluation of High Molecular Weight Multifunctional Acid-degradable Polymeric Drug Carriers with Structurally Different Ketals Rajesh A Shenoi, Srinivas Abbina, and Jayachandran N Kizhakkedathu Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01198 • Publication Date (Web): 17 Oct 2016 Downloaded from http://pubs.acs.org on October 26, 2016

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In vivo biological evaluation of high molecular weight multifunctional acid-degradable polymeric drug carriers with structurally different ketals Rajesh A. Shenoi1, Srinivas Abbina1 and Jayachandran N. Kizhakkedathu 1,2*

1

Centre for Blood Research and Department of Pathology & Laboratory Medicine, University of British Columbia, Vancouver BC, Canada V6T 1Z3. 2

Department of Chemistry, University of British Columbia, Vancouver BC, Canada V6T 1Z3.

*Corresponding author: Dr. Jayachandran N. Kizhakkedathu Centre for Blood Research, Department of Pathology and Laboratory Medicine Department of Chemistry University of British Columbia Vancouver BC Canada V6T 1Z3 E-mail: [email protected]

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ABSTRACT Understanding the influence of degradable chemical moieties on in vivo degradation, tissue distribution and excretion is critical for the design of novel biodegradable drug carriers. Polyketals have recently emerged as a promising therapeutic delivery platform due to their ability to degrade under mild acidic intracellular compartments and generation of non-toxic degradation products. However, the effect of chemical structure of the ketal groups on the in vivo degradation, biodistribution and pharmacokinetics of water soluble ketal-containing polymers has not been explored. In the present work, we synthesized high molecular weight, water soluble biodegradable hyperbranched polyglycerols (BHPGs) through the incorporation of structurally different ketal groups into the main chain of highly biocompatible polyglycerols. BHPGs showed pH and ketal group structure dependent degradation in buffer solutions. When the polymers were intravenously administered in mice, a strong dependence of in vivo degradation, biodistribution, and clearance on the ketal group structure was observed. All the BHPGs demonstrated degradation and clearance in vivo, with minimal tissue accumulation. Interestingly, an unanticipated degradation behavior of BHPGs with structurally different ketal groups was observed in vivo in comparison to their degradation in buffer solutions. BHPGs with cyclohexyl ketal (CHK) and cyclopentyl ketal (CPK) groups degraded much faster and were cleared from circulation much rapidly, while BHPG with glycerol hydroxy butanone ketal (GHBK) group degraded at a much slower rate and exhibited similar plasma half-life as that of non-degradable HPG. BHPG-GHBK also showed significantly lower tissue accumulation than non-degradable HPG after 30 days of administration. The difference in vivo degradation may be attributed to the difference in hydrophobic characteristics of different ketal containing polymers which may change their interaction with proteins and cells in vivo. This is the first study that demonstrates the influence of chemical structure of ketal groups on in vivo degradation and circulation profile of polymers, and through proper surface modifications, these polymers would be useful as multifunctional drug carriers. 2 ACS Paragon Plus Environment

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Key Words: Biodegradable polymers, Polyketal, In vivo degradation, Long circulating degradable polymers, Tissue accumulation, Drug carrier polymers

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1. INTRODUCTION The development of novel biodegradable, multifunctional and biocompatible polymers with controllable degradation and ability to respond to physiological and intracellular stimuli is an area of extensive research in drug delivery and tissue engineering.1-9 When used as drug delivery vehicles, high molecular weight, water soluble polymers are retained in the blood compartment for longer periods without elimination resulting in better therapeutic effect and improved targeting to the desired tissues such as tumor. However, high molecular weight polymers without any biodegradable linkages in their structure tend to accumulate in the vital organs that may impart long-term toxicity to the tissues. Hence incorporation of multiple biodegradable linkages in the main chain is desirable for biomedical applications wherein complete degradation of the polymer could be achieved after the intended function and the products are easily excreted from the body. Polyesters are the most widely explored biodegradable systems, but majority of the in vivo degradation studies of these polymers have focussed on subcutaneous implant materials.10-13 There is limited information available on the in vivo degradation of intravenously administered water soluble biodegradable polymers. Additionally, there is search for alternate biodegradable systems due to the increasing concerns over large quantities of acidic degradation products resulting from the hydrolytic degradation of polyesters as this can result in local pH reduction and tissue inflammation.14 The design and development of polymeric biomaterials requires a fundamental understanding of how the chemical structure and polymer architecture influence the in vivo degradation, organ distribution and cellular and tissue responses. The effect of polymer architecture on in vivo behavior has been demonstrated for polyesters by Nasongkla et al.; the polymers with cyclic structure exhibited longer blood circulation time and higher organ accumulation than those with linear structure of comparable molecular weight.15,

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Domb and Nudelman showed that the in vivo elimination of aliphatic

anhydrides depended on the chain length of the diacid monomers.10 Though there was little effect of 4 ACS Paragon Plus Environment

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the chain length on the in vivo degradation rate, the elimination rates were different due to the difference in the solubility of the degradation products. Another approach to control the degradation rate of polyesters is through copolymerization or physical blending with other polymers.12, 13 However, there is still limited information on how the in vivo degradation and clearance from the body are affected by the chemical structure of the degradable moiety in the polymers and their degradation products. Another challenge in the design of biodegradable polymeric drug carriers is that the in vitro degradation and drug release profiles usually could not be reciprocated in the in vivo environment when the material encounters complex biological media and cells. Recently ketal-containing polymers have been developed as a promising biodegradable drug delivery platform.17, 18 Polyketals are stable in the extracellular environment, but undergo hydrolytic degradation in the mildly acidic intracellular compartments. Moreover, the degradation products of these polymers are ketones and diols that are much less toxic when compared to the acidic degradation products of polyesters. These features make them attractive for intracellular delivery of proteins, nucleic acids and drugs as well as in targeting the acidic microenvironment of tumor tissues. Polyketal-based microparticles have been studied as drug carriers for chemotherapeutic agents as well as antiinflammatory agents for acute inflammatory diseases.19-26 Degradation of ketal groups in the mildly acidic endosomal compartments has also been exploited for the delivery of nucleic acids.27-29 Most of these reports have focussed on low molecular weight, water-insoluble polymers with dimethyl ketal groups. The dimethyl ketal group undergoes hydrolysis very rapidly under acidic conditions and hence these polymers may not be suitable for long term drug delivery as soluble carriers. Yang et al. reported poly(cyclohexane-1,4-diyl-acetone dimethylene ketal (PCADK) with different hydrophobicity and showed that the degradation rate could be controlled by limiting the diffusion of water into these polymers.19 Another report by Schopf et al. described the accelerated in vivo degradation of a ketal group-containing polymeric MRI contrast agent by the introduction of carboxyl groups adjacent to the 5 ACS Paragon Plus Environment

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ketal groups.30 Molecular weight of the drug carrier plays a key role in controlling the degradation rate and release of the incorporated drug. In order to achieve sustained drug release over longer periods in the vasculature, it is critical to have drug carriers that exhibit prolonged blood circulation times with minimal tissue/organ accumulation. A compelling strategy to achieve this would be to design high molecular weight, water soluble polymers with structurally different ketal groups. Currently there is no report on the effect of ketal group structure on the in vivo degradation and tissue distribution of water soluble high molecular weight ketal-containing polymers. In this article, we describe the synthesis of water soluble, multifunctional and high molecular weight biodegradable polymers with different ketal groups in the main chain in an attempt to control the degradation rate. These biodegradable hyperbranched polyglycerols (BHPGs) were synthesized through the incorporation of structurally different ketal groups into the main chain of highly biocompatible hyperbranched polyglycerols.31, 32 We hypothesized that the high molecular weight of these polymers would result in a longer blood residence time and the presence of structurally different ketal groups would enable to tune their degradation rates in vivo. The effect of ketal group structure on the in vivo degradation, biodistribution and excretion of the polymers upon intravenous administration in mice was investigated. 2. EXPERIMENTAL SECTION 2.1. Materials All the reagents and chemicals were purchased from Sigma-Aldrich, Canada (Oakville, Ontario) and used without further purification unless mentioned. Glycidol was purified by distillation under reduced pressure before use and stored over molecular sieves at 4 °C. The ketal monomers, 2-{1-methyl-1-[2(oxiran-2-yl methoxy) ethoxy] ethoxy} ethanol (CPKM), 2-(1-(2-(oxiran-2-ylmethoxy) ethoxy) cyclohexyloxy) ethanol (CHKM) and 2-(2-methyl-4-((oxiran-2-yl methoxy)methyl)-1,3-dioxolan-26 ACS Paragon Plus Environment

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yl)ethanol (GHBKM) were synthesized as per our previous publication.33 Tritiated methyl iodide was purchased from ARC Radiochemical (St. Louis. MO) as solution in toluene and was used directly after dilution in dimethylsulfoxide. 2.2. General procedure for the synthesis of high molecular weight biodegradable polymers A typical polymerization procedure for the synthesis of biodegradable hyperbranched polyglycerol with CPK monomer (BHPG-CPK) is as follows. Polymerization was carried out under argon atmosphere in a three-necked glass reactor equipped with a mechanical stirrer and a syringe pump. The initiator, trimethylol propane (TMP, 20 mg) was added to the flask and stirred with potassium methylate (0.040 mL) for 30 minutes. Excess methanol was removed under vacuum for 5 h. The temperature was increased to 105 °C and a mixture of glycidol (1.2 mL, 18.3 mmol) and 2-{1-methyl1-[2-(oxiran-2-yl methoxy) ethoxy] ethoxy} ethanol (CPKM) monomer (4.5 g, 18.3 mmol) was added slowly using a syringe pump. After complete addition, the reaction mixture was stirred for additional 12 h. To the same reaction pot, the glycidol (7 mL, 106.8 mmol) was added using syringe pump. After complete monomer addition, the reaction continued for additional 4 h. The resulting polymer was dissolved in methanol, precipitated thrice from acetone to remove the unreacted monomers and dried under vacuum. The other BHPGs were synthesized using similar procedure by changing the monomer to initiator ratio and using CHKM and GHBKM monomers instead of CPKM. The BHPG polymers were fractionally precipitated in acetone after dissolving in methanol.34 The polymer fractions with number average molecular weight (Mn) of 100 kDa and a polydispersity of less than 1.5 were used in the present work. 2.3. Polymer characterization 1

H spectra of the polymers were recorded in D2O on Bruker Avance 300 MHz NMR spectrometer.

Inverse gated (IG)

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C NMR spectra of the polymers were recorded in deuterated methanol (CD3OD) 7 ACS Paragon Plus Environment

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with a relaxation delay of 6 sec. The degree of branching was calculated as per the reported procedure from the equation, DB = 2D/ (2D + L), where D and L represent the intensities of the signals corresponding to the dendritic and linear units respectively.35 Absolute molecular weights of the polymers were determined by Gel Permeation Chromatography (GPC) on a Waters 2695 separation module fitted with a DAWN HELEOS II multi angle laser light scattering (MALLS) detector coupled with Optilab T-rEX refractive index detector, both from Wyatt Technology. Hydrodynamic radii (Rh) of the polymers were measured by quasi elastic light scattering (QELS) using Wyatt Internal QELS instrument (angle of measurement, 99.9°) GPC analysis was performed using Waters ultrahydrogel columns (guard, linear and 120) and 0.1 N NaNO3 at pH 8.5 (10 mM phosphate buffer) as the mobile phase. The dn/dc measured for the BHPGs was 0.12 similar to that of HPG. 2.4. Degradation of the polymers in buffer solutions pH dependent degradation of the BHPGs was studied by NMR and GPC analyses. For NMR analysis, the BHPG polymers were dissolved in deuterated buffer solutions of different pH values (prepared by dissolving the required amount of salts in deuterium oxide) and the degradation was monitored by collecting 1H NMR spectrum at different time intervals. The following buffer solutions were used for the study, potassium hydrogen phthalate/NaOH (pH 4.1), and potassium dihydrogen phosphate and NaOH (pH 5.5 and 7.4). In a typical experiment to measure the degradation kinetics, 12-15 mg of the BHPG was dissolved in 0.6 mL of the respective buffer solution and the NMR spectra were recorded at various time intervals. The percentage of polymer degradation was calculated from the intensities of the peaks corresponding to the ketal group in BHPG and ketone groups formed due to its degradation. For GPC analysis, two different methods were used. To determine the molecular weight after complete degradation, the BHPGs were stirred with a buffer solution of pH 1.1 for 24 h. The solution was neutralized with sodium hydroxide and the resulting solution analyzed by GPC-MALLS in aqueous 0.1 8 ACS Paragon Plus Environment

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N NaNO3 at pH 8.5. The kinetics of polymer degradation was studied by incubating the polymer samples in phosphate buffered saline (pH 7.4) at 37 °C and measuring the molecular weight of the aliquots at different time intervals. The molecular weight and polydispersity of the degraded polymers were compared with the values before polymer degradation. 2.5. Radiolabeling of BHPGs Radiolabeling of the BHPGs was performed by following our previous published reports.36 The labeling was achieved by the conversion of a portion of hydroxyl groups (~1%) to methoxy groups using tritiated methyl iodide. In a typical experiment, one hundred milligrams of the polymer was dissolved in 2 mL of 1-methyl-2-pyrrolidinone (NMP) and approximately 5% of the hydroxyl groups were deprotonated using sodium hydride. A calculated amount of tritiated methyl iodide (toluene solution) dissolved in DMSO was added to this solution so as to achieve methylation of approximately 1% of the hydroxyl groups. The reaction mixture was stirred at room temperature for 15 h, 10 mL of phosphate buffer (pH 9) was added and the labelled polymer was purified by dialysis against phosphate buffer (pH 9) using MWCO 1000 dialysis membrane until the dialyzate retained only low amounts of radioactivity; this took approximately 48 h. The polymer solution was then filtered through 0.2 µm syringe filter and the polymer weight was determined from the total volume and the polymer concentration (determined by freeze-drying a known volume of the solution). The tritiated polymer solution for animal study experimentation was prepared by the addition of appropriate amount of NaCl to achieve the desired osmolarity and the specific activity measured by scintillation counting. 2.6. Biodistribution studies The animal studies were conducted at the Experimental Therapeutics Laboratory of the British Columbia Cancer Research Centre, Vancouver, Canada. The protocol was reviewed and approved by the Institutional Animal Care Committee (IACC) at University of British Columbia. Female Balb/C 9 ACS Paragon Plus Environment

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mice (6-8 weeks, 20-26 g) were randomly grouped (three mice per group) based on their body weights for the study at different time points. The animals were injected intravenously (bolus) via lateral tail vein with the polymer solution at the prescribed dose of 10 mg/Kg, the injected volume being 200 µL per 20 g mouse. At different time points (0.5, 1, 2, 4, 24, 48 and 144 h), the mice were terminated by CO2 inhalation and blood was collected by cardiac puncture. For the BHPG-GHBK polymer, two additional time points at 15 days and 30 days were included in order to assess the long-term degradation of the polymer. Plasma was separated by centrifuging the blood samples at 2000 rpm for 10 min. Aliquots of plasma were analyzed for their radioactivity by scintillation counting. Upon termination, major organs such as liver, spleen, kidneys, heart and lungs were removed from the animals, weighed and processed for scintillation counting. Livers were made into a 30% homogenate in a known amount of water using a polytron tissue homogenizer. Aliquots (in triplicates) of 200 µL of the homogenate were transferred to scintillation vials for counting. All other organs were dissolved in 500 µL Solvable. The vials were incubated at 50 °C overnight, then cooled prior to addition of 50 µL 200 mM EDTA, 25 µL 10 M HCl and 200 µL 30% H2O2. This mixture was incubated at room temperature for 1 h prior to addition of 5 mL scintillation cocktail and radioactivity in the samples were measured by scintillation counting. Excretion of the polymers was studied by measuring the radioactivity in urine and feces with time. For this, mice at the 144 h time point were housed in metabolic cages and urine and feces were collected as pooled samples at different time points. Aliquots of urine were analyzed for radioactivity by scintillation counting. Feces were made into 10% homogenate into a known amount of water and the radioactivity was measured by scintillation counting. 2.7. Stability of BHPGs in serum

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Serum stability of BHPGs was studied by incubation of 200 µL of the polymer in 2 mL of fresh human serum. The polymer/serum mixture was then transferred to a dialysis cassette of 10 kDa cut off and dialyzed against PBS (pH 7.4) at 37 °C. At various time points, an aliquot (50 µL) of the solution from the dialysis bag was taken out and the radioactivity was measured by scintillation counting. The decrease in radioactivity was correlated with the degradation of the polymer. 2.8. Statistical analysis Unpaired, two-tailed t-tests were used for the statistical analysis of the data (Student t-test with MS Excel using two samples with two equal variances method). Values were considered significant if p < 0.05. All data are presented as mean ± standard deviation (SD) unless otherwise specified. 3. RESULTS AND DISCUSSION The precise control of in vivo degradation of functional polymers is required for applications ranging from drug delivery to tissue engineering. In contrast to the wealth of information on polymers as subcutaneous implantable materials,10-13 there is limited data available on the in vivo degradation of water soluble synthetic polymers. Such knowledge is important in the design of drug carriers with welldefined blood circulation time and low accumulation in vital organs. Ketal groups incorporated within the main chain of the polymer that could be degraded under acidic conditions encountered in intracellular compartments would be an attractive strategy to control the polymer degradation in vivo. In an earlier work, we have synthesized high molecular weight polymers containing dimethyl ketal group that degraded in vivo and showed a plasma circulation half-life of about 3 h with minimal tissue accumulation.34 Though this polymer may be suitable for rapid short term drug delivery, alternate structures are required for sustained retention of drug carriers within the vasculature. Thus in the present work, we sought to examine if the incorporation of structurally different ketal groups into the backbone of highly functional hyperbranched polyglycerols (HPGs) would impart different degradation 11 ACS Paragon Plus Environment

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rates in vivo. Since the in vitro cell toxicity of ketal containing BHPGs have been investigated thoroughly in our previous work,26,33,34 we concentrated on their in vivo degradation behaviour in the current manuscript. We anticipate that these BHPGs would have similar cell compatibility profile as reported previously 26,33,34. 3.1. Synthesis of water soluble functional high molecular weight BHPG polymers with structurally different ketal groups Structurally different ketal groups were introduced within the HPG main chain by the anionic ring opening multi-branching (co)polymerization of glycidol with AB2 type epoxide monomers with ketal groups such as cyclopentyl ketal (CPK), cyclohexyl ketal (CHK) and glycerol-hydroxybutanone ketal (GHBK) (Figure 1). A core-first methodology with slow monomer addition was employed for the synthesis of these biodegradable hyperbranched polyglycerols (BHPGs) in a single pot reaction at 105110 °C. This protocol resulted in improved reactivity of the ketal monomers and a more uniform incorporation of ketal groups within the polymers. Initially, a degradable polymer core was synthesized using 1:1 molar ratio of glycidol and the entire amount of the ketal monomer, and the polymerization was further continued in the same pot by the addition of the remaining amount of glycidol. The complete consumption of the ketal monomer was further confirmed by 1H NMR spectroscopy before adding glycidol to the reaction mixture. Since the bulk polymerization resulted in BHPGs of high polydispersity, the high molecular weight polymers (∼100 kDa) with low polydispersity for in vivo studies were obtained by using a fractional precipitation technique.34 The 100 kDa BHPG polymer fractions were obtained at methanol: acetone ratio of 1:1.8. The characteristics of these polymers are listed in Table 1 and the GPC profiles are shown in supplementary information (Figure S1). All the three BHPGs (BHPG-CPK, BHPG-CHK, and BHPG-GHBK) exhibited low polydispersity (less than 1.5), small hydrodynamic radius (5.3-5.8 nm) and high water solubility. The ketal content in the BHPGs was in the range of 12 to 15 mole % as determined by 1H NMR spectroscopy (supplementary 12 ACS Paragon Plus Environment

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information; Figures S2-S4), and the values were close to the feed composition indicating a higher reactivity of the monomers at elevated temperatures. The hyperbranched structure of the polymers was confirmed by inverse-gated

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C NMR spectroscopy (supplementary information, Figures S5-S7) and

the degree of branching was in the range of 0.56-0.59 for the polymers, the values were close to those reported for HPGs.35 3.2. pH dependent degradation of the BHPG polymers in buffer solutions in vitro The potential utility of the synthesized high molecular weight functional polymers for intracellular drug delivery requires understanding of their degradation behavior under physiological and intracellular conditions. So we investigated the degradation kinetics of the polymers in deuterated buffer solutions at pH values 5.5 and 7.4 (corresponding to the endosomal and extracellular conditions respectively) and 37 °C using 1H NMR spectroscopy. The percentage of degradation was calculated from the signals corresponding to the ketal groups on the polymer and the ketone group resulting from the degradation (Figures 2A, 2C and 2E). The hydrolysis half-life of the polymers was calculated by plotting concentration against time. Polymer degradation depended on the ketal group structure and the pH of the solution. On comparing the three polymers at pH 5.5, BHPG-CPK and BHPG-CHK showed very fast degradation with hydrolysis half-life of 0.23 h and 0.8 h respectively, while BHPG-GHBK that contains cyclic ketal groups did not exhibit any significant degradation during the short course of this experiment (Figure 2B). This could be attributed to the fact that in CPK and CHK, the ketal oxygen atoms form part of the chain (exocyclic), while in GHBK the ketal oxygen atoms form part of the ring (endocyclic). Hence the hydrolysis of GHBK requires cleavage of the endocyclic carbon-oxygen bonds that is disfavored due to entropy as well as enthalpy factors.37 But when the degradation was performed for longer periods, a slower degradation profile was observed for BHPG-GHBK, with approximately 20 % of polymer being degraded after 30 days. Polymer degradation was much slower at pH 7.4 for all the polymers, with hydrolysis half-life of 16.5 h and 54.5 h respectively for BHPG13 ACS Paragon Plus Environment

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CPK and BHPG-CHK polymers (Figure 2D). The BHPG-GHBK polymer did not show any significant degradation at this pH and was stable for more than 100 days. These results demonstrated that the degradation kinetics of BHPG polymers could be tuned by changing the pH of the solution and chemical structure of the ketal groups. Since the BHPG-GHBK did not show appreciable degradation at pH 5.5, its degradation was studied at pH 4.1; the pH encountered in the intracellular lysosomal compartment (Figure 2F). The degradation half-life was found to be 6 days at pH 4.1 and a complete degradation was observed after 24 days. The degradation half-life of BHPG-GHBK is intermediate between that reported earlier for polyketals based on PCADK.19 This may be attributed to the difference in the water solubility of the polymers. The degradation of a completely water soluble BHPG-GHBK is determined by the ketal group structure, while diffusion of water plays the major role in the degradation of poorly water soluble and predominantly hydrophobic PCADK.19 The data from the pH dependent degradation studies demonstrated a relatively higher stability of BHPG-CPK and BHPG-CHK polymers at the extracellular physiological pH, while much faster degradation occurs at the intracellular acidic environment, making them suitable for intracellular delivery of therapeutics. Though the BHPG-GHBK polymer degraded very slowly at pH 5.5, the much faster degradation at pH 4.1 could be explored for targeting the phagolysosomal compartments in the treatment of infectious diseases. BHPG-CHK and BHPG-CPK polymers could be suitable for rapid drug delivery, for example, during acute inflammatory events and BHPG-GHBK polymer could be useful for prolonged drug delivery in the treatment of chronic diseases as well as an implantable drug delivery system. When compared to previously reported PKHE polymers,33 the BHPG polymers showed slightly faster degradation presumably due to their higher water solubility. GPC profiles of the BHPGs before and after degradation are shown in the supplementary information (figure S8). As evident from the data, there is a shift in the refractive index (RI) peak of the polymers 14 ACS Paragon Plus Environment

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to higher elution volumes after degradation confirming the formation of low molecular weight polymer fragments. Polydispersity index of the polymers after degradation (1.9-2.7) was also higher than that of the original polymers (1.3-1.4), suggesting that the polymer fragments resulting from the degradation are of different molecular weights. Kinetics of degradation of BHPG-CPK and BHPG-CHK was also studied by determining the molecular weight with degradation time. The polymer samples were incubated in PBS at 37 °C and molecular weight of sample aliquots withdrawn at different time points was determined using GPCMALLS. A progressive decrease in molecular weight with time was observed for both BHPG-CPK and BHPG-CHK, with much faster decrease in molecular weight for BHPG-CPK (Figure 3 A). Molecular weight decreased from 100 kDa to 8 kDa in 5 days and 13 days for BHPG-CPK and BHPG-CHK respectively (Figure 3A). Approximately 90% reduction in the initial molecular weight was observed over this time interval (Figure 3B). The degradation half-life, the time required for polymer molecular weight to decrease to half of its initial value, was about 4 h and 48 h for BHPG-CPK and BHPG-CHK respectively. The degradation products of the BHPGs are ketones that are neutral and non-toxic when compared to the acidic degradation products resulting from polyesters. Thus cyclohexanone, cyclopentanone and 4hydroxy-2-butanone are the degradation products from BHPG-CHK, BHPG-CPK and BHPG-GHBK respectively. The toxicity, metabolism and excretion of these ketones are well-documented in the literature. 38-41 These molecules were found to be reduced to the corresponding secondary alcohols and excreted as glucuronic acid conjugates when administered via different routes in animals as well as in humans, without causing any acute or chronic toxicity38-41. Thus we anticipate that BHPGs with different ketal structures to be non-toxic in vivo. 3.3. Degradation of BHPG polymers in vivo 3.3.1. Plasma circulation of BHPGs with structurally different ketal groups 15 ACS Paragon Plus Environment

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We next investigated the plasma circulation behavior of the BHPGs with structurally different ketal groups after intravenous administration of tritium-labeled polymers in female Balb/c mice. At different time points, blood was withdrawn from the animals and the amount of radioactive polymer was measured by scintillation counting. The structure of the ketal groups within the polymer strongly influenced their plasma circulation; BHPG-GHBK showed almost similar circulation profile as that of the non-degradable HPG, while BHPG-CHK and BHPG-CPK were rapidly cleared from plasma within the first few minutes (Figure 4). Thus only 34% and 2.4% injected dose per gram (ID/g) of the polymers were observed in the plasma after 30 minutes for BHPG-CPK and BHPG-CHK respectively. This data suggests a faster degradation of BHPG polymers in vivo in comparison to that observed in buffer solutions. This result is unanticipated as BHPGs with dimethyl ketal as degradation points showed a relatively good correlation between the in vitro and in vivo degradation half-lives.34 We attribute this observation to the differences in the hydrophobicity of the randomly distributed ketal groups within the BHPGs which in turn affects their interaction with the blood components. Hydrophobicity of the ketal groups was found to follow the order CHK > CPK > GHBK> dimethyl ketal.33 The higher hydrophobic character of the cyclopentyl and cyclohexyl groups in comparison to the dimethyl groups might have resulted in stronger interaction of the BHPG-CPK and BHPG-CHK polymers with the plasma proteins leading to opsonisation and eventually much rapid clearance from circulation. However, our previous studies33 confirmed that there was no hemotoxicity associated with similar class of polymers with higher ketal content (no complement or platelet activation, no influence on blood coagulation and no cell toxicity) suggesting that the rapid clearance of BHPG-CPK and BHPG-CHK polymers in mice may not be due to the toxic side effects. Our argument on the influence of hydrophobic character on rapid elimination is supported by the published work on the fate of metallic nanoparticles in circulation42-44 and this may apply as well for the BHPG polymers. We have

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also recently shown that hydrophobically modified non-degradable HPGs showed rapid elimination from circulation compared to highly hydrophilic HPGs.45 Plasma circulation half-life of BHPGs was calculated by analysis of the data using the twocompartment open model.46 The 100 kDa BHPG-GHBK polymer showed plasma circulation half-life (40 h) (N = 3) close to that of non-degradable HPG of comparable molecular weight. Due to the faster clearance of the BHPG-CPK and BHPG-CHK polymers from circulation, we were not able to determine their plasma circulation half-life. The results from the study reveal two aspects. Firstly, the circulation time of biodegradable HPGs in vivo can be altered by changing the chemistry of the incorporated ketal groups in the main chain. Secondly, in addition to the chemical structure, factors such as hydrophobicity of the polymer may play a role in the elimination of the polymer from circulation. The study also reveals that polymeric nanoparticles may behave quite differently when they encounter biological fluids in vivo and the degradation rate could not always be correlated with that observed in vitro in buffer solutions. In the reported in vivo studies of polyketals using drug loaded polymers, comparison of in vitro and in vivo degradation rates of the polymers alone was not made. 1923

But these studies have demonstrated that the plasma circulation time of the drug could be

significantly prolonged by incorporation into polyketal microparticles or micelles.19-23 We anticipate that appropriate surface modification of BHPG polymers such as shielding of the hydrophobic groups and designing ketal groups which do not alter the overall physical characteristics of the polymers may allow the improvement in the blood circulation time of the rapidly degrading soluble ketal polymers. Previous studies have shown that protecting the hydrophobic groups in hyperbranched polyglycerol with polyethylene glycol chains could minimize the polymer-blood protein interaction thereby prolonging their plasma residence time. 45, 47, 48 3.3.2. Organ distribution of BHPGs with structurally different ketal groups

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The measurement of plasma circulation time of BHPG polymers demonstrated that both BHPG-CPK and BHPG-CHK polymers had faster elimination from circulation, and BHPG-GHBK showed prolonged residence in the blood compartment. Based on this data, one could anticipate a higher tissue accumulation for short circulating BHPG polymers if these polymers were not degraded within the body. In the case of BHPG-GHBK polymer a gradual increase in organ accumulation is anticipated as the polymer slowly exits from circulation. To investigate this, tissue distribution of tritium labeled BHPG polymers after intravenous injection at different time intervals was measured. As shown in Figure 5, both BHPG-CPK and BHPG-CHK polymers showed negligible accumulation ( BHPG-CPK > BHPG-GHBK, that was correlating with the in vivo blood circulation and clearance behavior of these polymers. A comparison of reduction in radioactivity of BHPG-CPK polymer between the serum and control PBS buffer is shown in Figure 7B; the degradation rate was slightly higher in serum at longer time intervals compared to that in buffer solution. However, this difference is not large enough to completely explain the very short plasma residence time of BHPG-CHK and BHPG-CPK. As explained earlier, the polymers did not activate complement system, a major source of opsonisation.50 Thus further studies are needed to understand the protein and vascular cell interactions of these polymers to explain the unanticipated behaviour of BHPG-CHK and BHPG-CPK polymers; these studies are in progress. 4. CONCLUSIONS We described the design and synthesis of high molecular weight water soluble multifunctional drug carriers that contain acid-sensitive ketal groups with different chemical structures. The BHPGs showed much faster degradation rates under mildly acidic conditions and the degradation rate was strongly dependent on the ketal group structure. When the polymers were administered intravenously in mice, rapid degradation and short plasma residence time were observed for the BHPGs with cyclopentyl and cyclohexyl ketal groups in comparison to their in vitro degradation. This unanticipated observation may be either due to rapid degradation of the polymers in the complex in vivo environment or the interaction of the hydrophobic ketal groups with the plasma proteins. BHPG with slow degrading GHBK groups showed prolonged circulation time similar to that of non-degradable HPG, but exhibited much lower tissue accumulation compared to non-degradable HPG one month post-administration making it suitable as prolonged and sustained degradable drug carrier. To the best of our knowledge, this is the first report on the investigation of the influence of ketal group chemical structure on the in vivo behavior of polyketals. Considerable opportunities exist to further alter the biodegradation profiles of

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these polyketal based biodegradable HPGs for their use as drug carriers and tissue engineering scaffolds, and such studies are in progress. ASSOCIATED CONTENT Supporting information Additional NMR spectra of representative degradable polymers (1H and 13C) and GPC-MALLS traces of polymers and degraded products (PDF) are given. This material is free of charge via the internet at http://www.pubs.acs.org. Acknowledgements The authors acknowledge the funding by the Canadian Institutes of Health Research (CIHR) and the Natural Sciences and Engineering Research Council (NSERC) of Canada. Authors thank Nancy Dos Santos for assistance with animal study at the IDP of the BC Cancer Research agency. The authors thank the LMB Macromolecular Hub at the UBC Center for Blood Research for the use of their research facilities. These facilities are supported in part by grants from the Canada Foundation for Innovation (CFI) and British Columbia Knowledge Development Fund (BCKDF). JNK holds a Career Investigator Scholar award from the Michael Smith Foundation for Health Research.

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GRAPHIC FOR MANUSCRIPT

Figure 1. Chemical structures of biodegradable HPGs (BHPGs) with structurally different ketal groups synthesized by the copolymerization of glycidol and ketal group-containing epoxide monomers.

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Figure 2. Degradation kinetics of BHPGs with structurally different ketal groups at 37 °C studied by 1

H NMR. The polymers were dissolved in deuterated buffers of different pH values and the percentage

of degradation was calculated from the ratio of the intensities of peaks corresponding to the ketal and ketone groups. Time- dependent NMR spectra for (A) BHPG-CHK at pH 5.5; (C) BHPG-CPK at pH 7.4; (E) BHPG-GHBK at pH 4.1 and degradation kinetics for BHPGs at (B) pH 5.5; (D) pH 7.4 and (F) pH 4.1 are shown. 24 ACS Paragon Plus Environment

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Figure 3. Degradation kinetics of BHPG-CPK and BHPG-CHK studied by GPC-MALLS. The polymers were incubated in PBS at 37 °C and molecular weight of aliquots withdrawn at different time points was determined. (A) Decrease in molecular weight with time; (B) Percentage decrease in molecular weight with time. The molecular weight decreased from about 100 kDa to 8 kDa (about 90% reduction) in 5 days for BHPG-CPK and 13 days for BHPG-CHK.

Figure 4. Plasma circulation profiles of BHPGs with structurally different ketal groups and nondegradable HPG of comparable molecular weight after intravenous administration of tritium-labeled polymers in female Balb/c mice (N=3). BHPG-CPK and BHPG-CHK showed rapid blood clearance, while BHPG-GHBK exhibited similar plasma circulation profile as that of non-degradable HPG. 25 ACS Paragon Plus Environment

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Figure 5. Tissue distribution of tritium-labeled high molecular weight BHPGs and non-degradable HPG after intravenous administration in female Balb/c mice (N=3); (A) Liver; (B) Spleen; (C) Kidneys; (D) Lungs; (E) Heart. BHPG-CPK and BHPG-CHK showed negligible accumulation (CPK >GHBK pH and hydrophobicity dependent 90–110 kDa

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