Thermoresponsive Random Poly(ether urethanes) with ......ring with CaH 2 in anhydrous dichloromethane (DCM), followed by ﬁ ltration and solvent removal in vacuo. Anhydrous DCM was

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aromatic diisocyanates [ 5–7 ] and/or organotin catalysts, [ 8–10 ] which are essential for their synthesis but may not be adequate in the fi nal products. [ 11,12 ] In order to address this problem, aromatic diisocyanates have been replaced by the more biocompatible aliphatic diisocyanates. [ 13–15 ] Generally, this is circumvented by the use of harsher poly-merization conditions or higher catalyst loadings to obtain high-molecular-weight products. [ 16,17 ] With the discovery of various organocatalysts that can serve as effective

A new class of thermoresponsive random polyurethanes is successfully synthesized and characterized. Poly(ethylene glycol) diol ( M n = 1500 Da) and 2,2-dimethylolpropionic acid are reacted with isophorone diisocyanate in the presence of methane sulfonic acid catalyst. It is found that these polyurethanes are thermoresponsive in aqueous media and manifest a lower critical solution temperature (LCST) that can be easily tuned from 30 °C to 70 °C by increasing the poly(ethylene glycol) content. Their sharp LCST transitions make these random poly-urethanes ideal candidates for stimuli-respon-sive drug delivery applications. To that end, the ability of these systems to effi ciently sequester doxorubicin (up to 36 wt%) by means of a soni-cation/dialysis method is successfully demon-strated. Additionally, it is also demonstrated that accelerated doxorubicin release kinetics from the nanoparticles can be attained above the LCST.

Thermoresponsive Random Poly(ether urethanes) with Tailorable LCSTs for Anticancer Drug Delivery

Haritz Sardon , * Jeremy P. K. Tan , Julian M. W. Chan , Daniele Mantione , David Mecerreyes , James L. Hedrick , * Yi Yan Yang *

Dr. H. Sardon, D. Mantione, Prof. D. Mecerreyes POLYMAT , University of the Basque Country UPV/EHU Joxe Mari Korta Center Avda. Tolosa 72, 20018 Donostia-San Sebastián , Spain E-mail: [email protected] Dr. J. P. K. Tan, Dr. Y. Y. Yang Institute of Bioengineering and Nanotechnology 31 Biopolis Way Singapore 138669 , Singapore E-mail: [email protected]

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Prof. J. M. W. Chan, Dr. J. L. Hedrick IBM Almaden Research Center 650 Harry Road, San Jose , CA 95120 , USA E-mail: [email protected] Prof. D. Mecerreyes Ikerbasque , Basque Foundation for Science E-48011 Bilbao , Spain

1. Introduction

Polyurethanes (PUs) have been considered as one of the most versatile classes of materials for a broad range of biomedical applications. [ 1–3 ] They have been used in heart valves, vascular grafts, catheters, prostheses, and other blood-contacting devices, on account of their versatility and biocompatibility. [ 4 ] However, their utilization in vivo has been limited by the need for toxic reagents such as

Early View Publication; these are NOT the final page numbers, use DOI for citation !!

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alternatives to tin-based catalysts, [ 8,18–20 ] new opportuni-ties for the preparation of PUs for biomedical applications have emerged. [ 8,21–23 ]

One of the most interesting classes of materials in nanomedicine is those giving response to different stimuli. [ 24,25 ] For instance, thermoresponsive materials have been investigated for tissue engineering, drug delivery of biosensing applications. [ 26,27 ] Among the different thermoresponsive materials, poly(N-isopro-pylacrylamide) (PNIPAM), polyvinylcaprolactam, and poly(ethylene glycol) are perhaps the most employed ones, but their lack of biodegrability limits their use. [ 28,29 ] The main reason for their broad utilization is the sharp thermal transition temperatures achieved with these polymers, together with the versatility of these polymers to easily tailor their thermal transition temperature by varying the macromolecular design.

It is well established that poly(ethylene glycol)-based polyether urethanes are considered the gold standard for biomedical applications because they are nontoxic, nonimmunogenic, and biocompatible. [ 30–32 ] In a recent report, Sun et al. [ 33 ] found that by adjusting the hydro-philic/hydrophobic balance of poly(ethylene glycol) (PEG) they could produce a series of temperature-sensitive drug delivery PU carriers. Nevertheless, the polymers showed very broad lower critical solution temperature (LCST) tran-sition (>10 °C) limiting their practical use as drug delivery systems. [ 34–36 ]

In this work, we describe the organocatalyzed synthesis of PUs with sharp and easily tunable LCSTs. Carboxylic acid functional groups were included in the poly(ether urethane) formulations due to their ability to seques-tering amine-containing drugs via strong ionic interac-tions between carboxylate and ammonium moieties. [ 37 ] These PUs self-assemble into nanoparticles with particles sizes in the range of 100–200 nm. The resulting random PUs were subsequently evaluated as temperature-sensi-tive drug delivery carriers that are capable of loading the model amphiphilic drug doxorubicin (DOX) with high effi ciency.

2. Experimental Section

2.1. Materials and Methods

PEG diol (PEG 1500 , M n = 1500 Da) and 2,2-bis(hydroxymethyl)-propionic acid (bis-MPA) were dried by azeotropic distillation in benzene. Isophorone diisocyanate (IPDI) was dried by stir-ring with CaH 2 in anhydrous dichloromethane (DCM), followed by fi ltration and solvent removal in vacuo. Anhydrous DCM was obtained by using a solvent purifi cation system from Innovative Technologies. Anhydrous methane sulfonic acid (MSA) was used as received. All other materials were purchased from Sigma–Aldrich and used as received.

1 H and proton-decoupled 13 C NMR spectra were obtained on a Bruker Avance 400 instrument. Gel permeation chromatography (GPC) was performed in tetrahydrofuran (THF) at 30 °C using a Waters chromatograph equipped with four 5 mm Waters col-umns (300 mm × 7.7 mm) connected in series with increasing pore size (100, 1000, 105, 106 Å). Fourier transform infrared spec-troscopy (FTIR) was used to confi rm the polymerization process: using a Nicolet Magna 560 spectrometer at a resolution of 2 cm −1 , and a total of 64 interferograms were signal-averaged. Samples were prepared by solution-casting the reaction mixture onto the KBr plate.

Dynamic light scattering (DLS) measurements were per-formed on Zetasizer 3000 HAS (Malvern, UK) equipped with a He–Ne 658 nm laser. The nanoparticle solution was fi ltered using a 0.45 μm poly(vinylidene fl uoride) (PVDF) fi lter before measure-ment. Each sample was measured three times and the average diameter of the particles, D h , was obtained.

Light transmission was determined using a UV–vis spectro-photometer Agilent 8453 equipped with a refrigerated circu-lator. The measurements were performed with a wavelength of 600 nm, and the sample solutions were equilibrated for 10 min before each measurement during the heating–cooling cycling. The cloud point temperature ( T cp ) could be determined from the onset of the sudden decrease in the transmittance of the polymer aqueous solution.

Transmission electron microscopy (TEM) was performed using a FEI Tecnai G 2 F20 electron microscope using an accelera-tion voltage of 200 keV. Polymeric sample (10 μL) was placed on a 200 mesh formvar/carbon-coated copper grid and allowed to dry for 1 min before the excess of the sample was removed using fi lter paper. Phosphotungstic acid (0.2% w/v) was added onto the grid for 1 min and excess was then removed using a fi lter paper.

Polymer cytotoxicity was evaluated using the (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay. Spe-cifi cally, liver carcinoma (HepG2) cells were seeded in a 96-well plate (10 000 cells per well) and incubated overnight in an incu-bator maintained at 37 °C and 5% CO 2 before being treated with the polymer at various concentrations for 48 h. Two tempera-tures were chosen to demonstrate the cytotoxicity of the polymer at below (32 °C) and above (37 °C) LCST to the cells. The cells were grown in Dulbecco’s modifi ed eagle medium (DMEM) media sup-plemented with 10% FBS and 1% penicillin–streptomycin. After 48 h treatment, the solution in each well was replaced with 20 μL of MTT (5 mg mL −1 in phosphate-buffered saline (PBS)) and 100 μL of DMEM and incubated for 4 h. MTT solution was care-fully removed and 150 μL of DMSO was added to dissolve the purple formazan crystals and measured using a TECAN micro-plate reader at 550 and 690 nm. The absorbance of the formazan crystal in each well was calculated as that at 550 nm deducted by that at 690 nm and cell viability was calculated as a percentage of absorbance against the untreated control sample.

2.2. Preparation of DOX-Loaded Micelles and Measurement of DOX Loading

DOX encapsulation was done through a sonication/dialysis method. DOX (5 mg) was neutralized with 3 mole excess of tri-ethylamine and dissolved in 1.5 mL of N , N -dimethylacetamide



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(DMAc). The DOX solution was mixed with polymeric solution (10 mg in 0.5 mL DMAc) by vortexing, and the mixture was added dropwise into 10 mL deionized (DI) water while being sonicated at 130 W for 2 min with a probe sonicator (Vibra Cell VCX 130). After this, the mixture was dialyzed against 1 L of DI water using a 1000 Da molecular weight cutoff membrane (Spectra/Por 7). The water was changed at 3, 6, 24 h and the sample was collected after 48 h and lyophilized. Each experiment was performed in triplicates. To determine the DOX loading level, a known amount of lyophilized DOX-loaded micelles was dissolved in 1 mL of dimethyl sulfoxide (DMSO) and the absorbance of the solution was measured using the UV–vis spectrometer at 480 nm. The DOX loading level was determined using the following formula:

DOX loading(wt%)

mass of DOX extracted from micellesmass of DOX loaded micelles initally used

100%

=

×

2.3. Synthesis of Random Polyurethanes

The acid-catalyzed polyaddition of diols with aliphatic diiso-cyanates for the synthesis of random PUs was performed in a glove box. Briefl y, fi ve different PUs were synthesized by varying the bis-MPA/PEG ratio. In a typical polymerization, the diols (1.00 equiv., 3.30 mmol) and IPDI (1.00 equiv., 3.30 mmol, 0.730 g) were dissolved in 14 mL of dry DCM (0.5 M of each monomer). (The relative proportion of the diols for the synthesis of dif-ferent PUs could be found in the Supporting Information). Sep-arately, MSA (0.050 equiv., 0.18 mmol, 0.025 g) was dissolved in 0.1 mL of dry DCM and added dropwise to the reaction mixture. The polymerization was carried out at 30 °C for 24 h and moni-tored by 1 H NMR. Table 1 shows the relative amount of reagents employed for the preparation of different PUs. Full experimental details are provided in the Supporting Information.

3. Results and Discussion

In the present work, we explore the use of MSA as a strong Brønsted acid for the synthesis of tin-free PUs con-taining pendant carboxylic acid moieties. This choice of catalyst was logical based on the mutual compat-ibility of the sulfonic acid and the carboxylic acid groups. The low-molecular-weight PEG diol ( M n = 1500 Da) was employed as a co-monomer to prevent protein adsorp-tion, and consequently improve the stability of nanopar-ticles in the physiological environment and prolong their blood circulation half-lives. In addition, it is well known that depending on the molecular design of PEG-containing polymers, the resulting polymers may be either insoluble in water with a suffi cient concentration of hydrophobic co-monomer or readily water soluble. Indeed, when the hydrophobic to hydrophilic components are properly incorporated in a polymer structure the polymer could become thermoresponsive. PUs are ideal polymers because depending on the monomeric units the biodegradability of the polymers can be tailored. [ 38–40 ]

3.1. Acid-Catalyzed Synthesis of PUs with Pendant Carboxylic Groups and PEG Moieties

Five different PUs were synthesized with various bis-MPA/PEG diol ratios, as shown in Table 1 . The polymerization was accomplished by dissolving equimolar amounts of IPDI and bis-MPA/PEG diols in DCM (2.0 M ) followed by the addition of 5 mol% of MSA (Scheme 1 ). The polymerization was monitored by 1 H NMR, FTIR, and GPC. In most cases, >98% conversion (by 1 H NMR) was observed within 24 h,



Table 1. Reagent ratios employed for the synthesis of the fi ve different PUs and the observed molecular weight distributions and conversions.

Sample bis-MPA [equiv. mmol −1 ]

PEG diol [equiv. mmol −1 ]

IPDI [equiv. mmol −1 ]

Conversion [mol%] ( 1 H NMR)

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PU-1 0/0 1.00/3.30 1.00/3.30 >98 30.1/1.3

PU-2 0.25/0.83 0.75/2.48 1.00/3.30 >98 22.6/1.4

PU-3 0.50/1.65 0.50/1.65 1.00/3.30 >98 19.5/1.6

PU-4 0.75/2.48 0.25/0.83 1.00/3.30 >98 17.6/1.8

PU-5 1.00/3.30 0.00 1.00/3.30 90 5300/2.2 a)

a) PU-5 was only partially soluble in THF.

Scheme 1. General MSA catalyzed synthesis of PUs from isophorone diisocyanate (IPDI), bis-MPA and PEG 1500 diol.


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demonstrating the effectiveness of MSA in catalyzing PU formation from carboxylic acid-containing co-monomers. Only in the case of PU-5, we were unable to achieve high conversion, probably due to the limited solubility of bis-MPA in DCM.

FTIR spectra of PU-3 shows that as the reaction pro-ceeded, there was an intensity decreased and ulti-mately complete disappearance of the isocyanate stretch (N C O) at 2265 cm −1 . Two new bands appeared (amide I at 1720 cm −1 , and amide II at 1550 cm −1 ), confi rming suc-cessful urethane linkage formation (Figure S1, Supporting Information). To further verify the polymerization process, 1 H NMR studies were also carried out (Figure S2–6, Sup-porting Information). As observed, when the reaction pro-ceeded, characteristic signals (δ = 3.10 ppm) of the meth-ylene protons proximal to the isocyanate decreased in intensity. At the same time, a new signal due to methylene protons linked to the urethane groups appeared at δ = 4.20 ppm, demonstrating the success of the reaction.

To further verify the formation of high-molecular-weight polymers, GPC analysis was performed (Figure S7, Supporting Information). Table 1 summarizes the molec-ular weight ( M w ) and dispersity index ( Ð ) values obtained for each PU. We observed, that the M w ranged between 17.6 and 30.1 kDa and Ð was between 1.3 and 1.8. When two diols with different levels of reactivity were used, a multimodal molar mass distribution was obtained. These values are in good agreement with those that we recently reported for MSA-catalyzed isocyanate-alcohol polymeri-zations. [ 41 ] The exception is PU-5, which was only partially soluble in THF and it was thus not possible to measure its molecular weight distribution.

The particle sizes of the polymeric samples were meas-ured below and above the LCST (Table 2 ). The TEM image displayed in Figure S8 (Supporting Information) showed that the size obtained from DLS agreed with that obtained from TEM. When measured below the LCST, the sizes are all below 40 nm. However, above the LCST, the particles aggregated together due to the collapse of the hydrophilic region. This exposed the hydrophobic domains and cause

aggregation of the individual micelles into larger aggre-gates of around 300 nm.

3.2. DOX Loading and Size of DOX-Loaded Nanoparticles

DOX was loaded into PU nanoparticles via sonication and dialysis. Triethylamine (TEA) was used to obtain the DOX free base in DMAc at 3:1 molar ratio of TEA to DOX prior to micelle formation and will be gotten rid off during the dialysis process. This neutralization process will increase the overall hydrophobicity of the drug therefore improving the loading content. Sonication was carried out to prevent the formation of large aggregates during encap-sulation. [ 31 ] As shown in Table 2 , PU-1 without carboxylic acid groups gave a very low DOX loading level (


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can be reduced to 26 °C when using nonstandard linear PEGs such as graft PEG (PEG methacrylates). [ 42 ]

Lutz [ 29,43 ] and others found that the key parameter that determines their solution properties is the balance between hydrophilic and hydrophobic moieties in the molecular structure of the polymers. Thus in the case of poly(ethylene glycol) methyl ether methacrylates, they found that using only two or three ethylene oxide repeating units the polymers were soluble at room temperature, because the balance between favorable polymer–water interactions and unfavorable hydro-phobic interactions is suffi cient to allow solubiliza-tion. Therefore, poly(ethylene glycol) methyl ether methacrylates showed LCSTs and this temperature is dependent on the overall hydrophilic–hydrophobic ratio. In addition, amide groups and acid groups, that are present in urethane linkages, are also able to strongly interact with water molecules inducing water solubility.

Similarly, with PEG-based PUs, the ether oxygen of PEG together with the amide bonds will form stabi-lizing H-bonds with water, whereas the hard segment of PUs brings about a competing hydrophobic effect. The LCST transition in aqueous solution reflects first of all a local structural transition involving water mol-ecules surrounding the polymer. Below the LCST, the polymer is hydrophilic and the water molecules are bound to its polar groups and to each other via hydrogen bonds. The PU may adopt an extended coil conformation. The relative magnitude of the hydro-phobic effect increases with temperature. At higher temperatures, water molecules are released in bulk, allowing associative interactions between the newly

exposed hydrophobic monomer units, collapsing the PU chains (Figure 2 ). Moreover, the LCST in this case also increased as we increased the hydrophilic ratio. The LCST values for all the polymers were decreased when aqueous phase was changed from DI water to the simulated physiological environment, 15 × 10 −3 M PBS (pH 7.4) 56 °C, 45 °C, 36 °C, and 26 °C for PU-1, PU-2, PU-3, and PU-4 respectively) ( Figure S9–S12, Sup-porting Information ) The introduction of salt shielded the charge, increasing the hydrophobicity of the poly-mers. It is worth noting that no hysteresis was observed when the sample was heated and subsequently cooled (Figure S13, Supporting Information).

3.4. In Vitro DOX Release

Since PU-3 and PU-4 have LCST measured in PBS that are below the body temperature, they can be used to deliver drugs through local injection. The hydrophilic shell would become hydrophobic leading to precipitation of the micelles thus promoting uptake by tumor tissues. In vitro release of DOX was conducted at 25 °C (below LCST) and 37 °C (above LCST) using DOX-loaded PU-3. As shown in Figure 3 , DOX release was temperature dependent and faster release was observed above the LCST. Above the LCST, the hydrophilic shell collapsed, leading to a deformed core/shell structure. This might in turn expose the DOX molecules to the solu-tion, accelerating their release. The nanoparticles after being injected in vivo could be passively targeted to the tumor via the enhanced permeability and retention (EPR) effect and localized heating can be conducted to trigger the release of drugs. This will greatly reduced the harmful side effects of these chemotherapy drugs.



Figure 1. Turbidity curves for PUs containing different PEG 1500 /bis-MPA ratios in DI water. The curves represent the transmittance of UV light (600 nm). The LCST temperature could be determined from the sudden decrease in the transmittance of the polymer aqueous solution.


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3.5. In Vitro Cytotoxicty

The cytotoxicity of PU-3 was evaluated against HepG2 cell line (Figure S14, Supporting Information). No cell death was induced by the polymer at either below (32 °C) or above (37 °C) the LCST demonstrating the potential of

these nanoparticles as a drug delivery carrier. These results are consistent with other results observed for PEG-based aliphatic PUs. [ 44 ]

4. Conclusion

New poly(ether urethanes) composed of PEG, bis-MPA, and IPDI were synthesized by methanesulfonic acid-catalyzed polyaddition. Successful polymerization was confi rmed by 1 H NMR, FTIR, and GPC. We found that these poly(ether urethanes) were thermoresponsive in aqueous media and manifested an LCST that can be easily tuned from 30 °C to 70 °C by increasing the poly(ethylene glycol) content. The versatile properties such as water solubility, ability to sequester a signifi cant amount of cargo, thermal sen-sitivity, and biodegradability make these PU-based mate-rials ideal for various biomedical applications. Here, we have demonstrated the ability of these systems to effi -ciently encapsulate amine-containing DOX through ionic interactions. Moreover, PU-3 and PU-4 nanoparticles with LCST values below the body temperature and temperature-responsive drug release are ideal candidates as injectable



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local drug delivery carriers. It is envisioned that this facile, metal-free synthesis of temperature-responsive PUs will provide a versatile and convenient platform for biomedical applications.

Supporting Information

Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgements: H.S. gratefully acknowledges fi nancial support from MINECO through project number FDI 16507 and Institute of Bioengineering and Nanotechnology (Biomedical Research Council, Agency for Science, Technology and Research, Singapore) is also acknowledged.

Received: April 27, 2015 ; Revised: June 2, 2015 ; Published online: ; DOI: 10.1002/marc.201500247

Keywords: drug-loading ; LCST ; nanoparticles ; organocatalysis ; polyurethanes

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Documents

Thermoresponsive Random Poly(ether urethanes) with ......ring with CaH 2 in anhydrous dichloromethane (DCM), followed by ﬁ ltration and solvent removal in vacuo. Anhydrous DCM was