Poly(anhydride-ester) Gemcitabine: Synthesis and Particle Engineering of a High Payload Hydrolysable Polymeric Drug for Cancer Therapy

Abstract

Gemcitabine (GMT) is a nucleoside analog used in the treatment of a variety of solid tumors. GMT was chemically modified with a hydrolysable linker, and subsequently incorporated into a poly(anhydride-ester) backbone via melt-polymerization, with the active antimetabolite GMT, thus, becoming the repeat unit that makes up this new material, a biodegradable polymer. Characterization of the structure of polymeric GMT (polyGMT) revealed the incorporation of an average 26 molecules of GMT per polymer chain, which corresponds to a drug loading of 58%w/w. The glass transition temperature of the formed polyGMT was determined to be 123 °C. PolyGMT was engineered into nanoparticles (NPs) using a dialysis-based method, with a resulting geometric diameter of 206 ± 38 nm. The particles are easily dispersible and stable in aqueous-based media, with a hydrodynamic diameter of 229 ± 28 nm. The prepared hydrolysable polyGMT NPs demonstrate ultra-long release profile due the hydrophobic nature of the linker, and as per characteristic erosion behavior of polymers with anhydride-ester bonds. Accelerated in vitro release studies demonstrate the recovery of free GMT upon hydrolysis, with biological activity as assessed by cytotoxicity assays performed in adenocarcinoma human alveolar basal epithelial (A549) and highly metastatic murine osteosarcoma (K7M2) cells lines. The characteristics of polyGMT, including its thermal properties and built in hydrolysable structure, are thus conducive for use in the preparation of drug delivery systems. Engineered structures prepared with polyGMT can maintain their morphology at ambient and physiologically relevant conditions, and free GMT is recovered as the anhydride and ester bonds are hydrolyzed. This work is innovative as for the first time we demonstrate the ability to polymerize GMT in a hydrolysable polymer structure, and engineer NPs of this polymeric chemotherapy. The synthetic strategy allows for tuning of the polymer hydrophobicity and thus potentialize its behavior, including degradation profile, by varying the linker chemistry. Such controlled release hydrolysable polymers with very high drug loading and controlled erosion profiles are relevant as they may offer new opportunities in drug delivery applications for the treatment of malignant neoplasms.

1. Introduction

Cancer is a global public health problem, with more than 1 in 3 adults expected to develop some form of the disease during their lifetime.[1] Toxicity,[2,3] drug resistance,[4] and low response rates[5] of chemotherapies continue to be significant issues in the fight against cancer. In spite of innovative treatments,[6] chemotherapy is still widely used to treat most types of cancers,[7] independent of their type, stage and molecular characteristics.[8]

Gemcitabine (GMT), an essential medicine for cancer according to the World Health Organization (WHO),[9,10] is broadly used alone or in combination with other therapies in the treatment of a variety of solid tumors, including pancreatic, breast, bladder, head, neck, thyroid, ovarian, non-small cell lung, and bone cancers.[11,12] GMT is a water-soluble chemotherapeutic, and as most other chemotherapeutics, it has poor pharmacokinetic (PK, e.g. very short half-life of 15 min)[11,13] and tumor biodistribution profiles (e.g. 0.5% of total dose in tumor upon systemic administration).[14]

Formulation of chemotherapeutics as nanomedicines[15,16] is a promising strategy for increasing their performance and safety.[17,18] Nano-sized drug carriers can be formulated to improve the PK profile of drugs, increase accumulation in the tumor site[19] and protect the active ingredient from degradation.[20]

However, in spite of positive outcomes in terms of improving efficacy and safety,[21] the majority of the formulations containing nanomaterials still facing challenges that include low drug loading and as a consequence, particularly for polymeric formulations, the use of a large ratios of inactive/active ingredient.[22,23] Drug loading in polymeric nanoparticles such those prepared with PLGA[24–28] or PLA[29–33] continue to be generally relatively low, usually representing less than 5% of active ingredient on a mass base.[22] In the case of gemcitabine, drug loading within PLGA nanoparticles is ca. 3%.[34,35] Given the broad potential applicability of the large number of polymer based nanocarriers that are currently in clinical trials,[36,37] an improved design strategy for this class of materials for the delivery of chemotherapy could provide an important new direction and help alleviate some of the existing challenges.

One potential way to overcome the high fraction of inactive agent inherent to synthetic (non-active) polymer-based nanocarriers is to incorporate the active pharmaceutical ingredient as a monomer/repeat unit of the polymer itself (polymeric drug), instead of encapsulating the drug within the core of a particle made with the polymer.[38,39] Polymeric drugs also represent significant gains in terms of loading compared to conjugation of drugs to polymeric backbones,[40] which have an additional challenge as the polymer backbone is still present after the release of the drug.[41,42] Even though several types of polymer chemistries could, in principle, be explored for the polymerization of drug monomers, polyanhydrides have several attractive features.[43] The interest in polyanhydrides ultimately derives from the fact that they possess intrinsic qualities that are attractive to be used in drug delivery systems, including biodegradability and low toxicity.[44,45] The combination of anhydrides and ester bonds[46] is also an exciting strategy, since ester bonds are also biodegradable[47,48] and demonstrate different stability profiles under hydrolytic conditions when compared to anhydride bonds.

The combination of ester and anhydride bonds has been used to prepare polymeric drugs of salicylic acid,[49,50] morphine,[51] antiseptics,[52] and antioxidants.[53] Interconnection of the active monomers via polymerization reaction was achieved in those cases by direct polymerization of the active ingredient or upon modification of the drug for cases when there was not sufficient functional groups on the drug molecule for polymerization. In this work, we demonstrate a synthetic strategy suitable for the incorporation of GMT at high loadings in a poly(anhydride-ester) backbone, and that such new materials (polyGMT) have appropriate thermophysical properties and hydrolysis profile suitable for their engineering as nano-sized drug delivery systems with potential applications in cancer therapy. We discuss the synthesis and characterization of the various steps leading to polyGMT and the preparation and engineering of nanoparticles of polyGMT using a dialysis-based method. We also discuss the biological activity of GMT recovered after hydrolysis in in vitro tumor models.

2. Materials and Methods

2.1. Materials

Gemcitabine hydrochloride (GMT.HCl) was purchased from Ark Pharm (Arlington Heights, IL, USA). Succinic anhydride (SA)₂, benzyl alcohol, 4-(dimethylamino)pyridine (DMAP), and acetic anhydride were obtained from VWR International (Radnor, PA, USA). Ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC), 2,5-dihydoxybenzoic acid (2,5-DHB), silica gel for chromatography (pore size 60 Å, 220–440 mesh particle size) were purchased from Sigma-Aldrich (St Louis, MO, USA). Anhydrous dichloromethane (DCM), dimethyl formamide (DMF), dimethyl sulfoxide (DMSO), diethyl ether, methanol (MeOH), tetrahydrofuran (THF) were purchased from VWR International (Radnor, PA, USA) and used as received, unless otherwise specified. HPLC grade solvents (acetonitrile, water) were obtained from EMD Millipore (Billerica, MA, USA). Spectra/Por regenerated cellulose (RC) membrane dialysis tubing was purchased from Spectrum Laboratories, Inc. (Rancho Dominguez, CA, USA). Deuterated DMSO (DMSO-d6) was purchased from Cambridge Isotope Laboratories (Tewksbury, MA, USA).

2.2. ¹H NMR and ¹³C NMR, MALDI-ToF and FTIR analysis

¹H and ¹³C NMR spectra were obtained using a Bruker 400 MHz spectrometer. Samples were dissolved (~5 mg/mL for ¹H NMR and ~50 mg/mL for ¹³C NMR) in deuterated dimethyl sulfoxide (DMSO-d6), which was used as an internal reference. Each spectrum was an average of 32 scans and 1024 scans, respectively, unless otherwise specified.

MALDI-ToF analyses were performed in a Voyager DE Pro MALDI ToF (AB Sciex, MA, USA), operating with a 337 nm Nitrogen Laser, using refractor mode. The analyzed spot was prepared as following: 0.5 μL of a 10 mg/mL sample solution in DMF was mixed with 50 μL of a 20 mg/mL matrix (DHB) solution in acetonitrile/water (1:1). An aliquot (1 μL) of this mixture was taken and spotted on the MALDI plate and left until dried.

ATR-Fourier transform infrared (FTIR) spectra were obtained using a Thermo-Fischer Nicolet IS20 spectrometer. Each spectrum was an average of 32 scans.

2.3. Differential Scanning Calorimetric Analysis (DSC) and Thermogravimetric Analysis (TGA)

DSC analyses were performed on a Perkin-Elmer system consisting of a Pyris 1 DSC analyzer with a TAC 7/DX instrument controller. Samples (10 mg) were heated at a rate of 10 °C/min, under nitrogen flow, with two-cycle minimum. Glass transition was calculated as half Cp extrapolation.[54] TGA analyses were performed on a Perkin-Elmer system (Pyris 1 TGA analyzer with a TAC 7/DX instrument controller).

2.4. Synthesis and Characterization of Poly(anhydride-ester)Gemcitabine

2.4.1. Synthesis of Gemcitabine-2SA-Bz (Compound 1 - Scheme 1)

Scheme 1.

Synthetic route for the preparation of acetylated gemcitabine (GMT-2SA-Acetyl). (Abbreviations: EDC: N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide, DMAP: 4-(Dimethylamino)pyridine, DMF: Dimethylformamide)

The 4-(benzyloxy)-4-oxobutanoic acid molecule (linker) was, firstly, obtained through the reaction of benzyl alcohol with (SA)₂ (Supporting Information - Fig S1). This linker molecule was, then, reacted with GMT.HCl (200 mg, 0.67 mmol - 1 equiv). The reagents were dissolved in 3 mL of anhydrous DMF, cooled down by an ice bath ( ~ 0 °C) and received the addition of DMAP (245 mg, 2.01 mmol - 3.3 equiv). Under vigorous stirring, the reaction mixture received the, dropwise, addition of a suspension of EDC (385 mg, 2.01 mmol - 3.3 equiv) in 1 mL of DMF. The reaction was stirred, in the absence of light, for 30 min on an ice bath and for additional 24 h at room temperature. The reaction solvent was, then, removed through high vacuum. The crude product was dissolved in DCM (150 mL) and submitted to liquid-liquid extraction. The undesired compounds were extracted with 10%w/w of NaHSO₄ solution (3 X 30 mL), 10%w/w of Na₂CO₃ solution (3 X 50 mL), and brine (1 X 30 mL). The organic phase was dried (MgSO₄), filtered, concentrated, and purified by liquid column chromatography on silica gel, eluting with DCM and gradually increasing the polarity to 10:90 MeOH:DCM, to give a yellowish oil, GMT-2SA-Bz. The compound was characterized by ¹H NMR, ¹³C NMR (Supporting Information - Fig S4–S5) and MALDI-ToF m/z (Da) - Calculated: 643.20 Obtained: [M+H]⁺ 645.89; [M+Na]⁺ 666.42.

2.4.2. Synthesis of Gemcitabine-2SA (Compound 2 - Scheme 1)

GMT-2SA-Bz (200 mg) was dissolved in THF and the catalyst 5%w/w Pd/C (2 mg) was added. The resulting suspension was placed in a hydrogenolysis apparatus. The vessel had its atmosphere removed through vacuum and replaced by pure H₂ (Airgas, Radnor, PA). The reaction was stirred overnight, in the absence of light, being the H₂ pressure kept at 14 psi. The catalyst was, then, removed through filtration (Whatman grade 50, GE, Buckinghamshire, UK) and the resulting filtrate had its solvent removed using a rotary evaporator (Rotavapor R-3, Büchi, Postfach, Switzerland), resulting in a transparent oil, GMT-2SA, which was used without further purification. The compound was characterized through ¹H NMR, ¹³C NMR (Supporting Information - Fig S6–S7) and MALDI-ToF m/z (Da) - Calculated: 453.10 Obtained: [M+H]⁺ 464.71; [M+Na]⁺ 486.87.

2.4.3. Synthesis of Gemcitabine-2SA-Acetyl (Compound 3 - Scheme 1)

GMT-2SA (200 mg) was reacted with a large excess of acetic anhydride (13 mL) in the absence of solvent. The reaction was stirred, in the absence of light, for 3 hours, until the disappearance of any solid in the reaction vessel. The excess of acetic anhydride and formed acetic acid were removed through high vacuum, resulting in a viscous yellowish oil, GMT-2SA-Acetyl. The compound was characterized through MALDI-ToF m/z (Da) - Calculated: 547.42 Obtained: [M+Na]⁺ 571.45; Dimer [M+Na]⁺ 992.75.

2.4.4. Polymerization Reaction

For the interconnection of the monomer molecules, a standard protocol for poly(anhydride) synthesis was utilized.[43] The acetylated monomer (GMT-2SA-Acetyl) was placed inside a reaction vessel, which was, in turn, placed in a silicon oil bath. A mechanical overhead stirrer (Fristaden Lab, NV, USA) was used to promote the mixing of the reactor content. The vessel was sealed and submitted to high vacuum (~2 mmHg). The temperature of the silicon oil bath was kept at 120 °C during the reaction and the stirring was set as 100 rpm. The reaction conditions were kept until the maximum viscosity (visually inspected) was reached (~30 min). The reaction vessel was cooled down to room temperature and the formed crude product was dissolved in DMF. The polymer was precipitated in cold (~ 0 °C) diethyl ether, twice. The isolated polymeric material was, then, dried under vacuum for 24 h, resulting in a beige solid, polyGMT. The final product was characterized through ¹³C NMR (Supporting Information - Fig S8), FTIR and GPC.

2.4.5. Poly(anhydride-ester)Gemcitabine Molecular Weight Analysis

Mass average molecular weight (Mw), number average molar weight (Mn), polydispersity index (PDI), solvated radius (Rh) and dn/dc were determined by gel permeation chromatography (GPC) on an Omnisec GPC system (Malvern-Panalytical, MA, USA). The system was equipped with four types of detectors: Refractive index (RI), UV-Vis, Right-angle light scattering (RALS), and Viscometer allowing the use of an universal calibration approach. The samples were analyzed using OMNISEC software (version 10). The polymer was dissolved in DMF and filtered through 0.22 μm PTFE syringe filters (VWR, Radnor, PA, USA) before analysis. The samples were resolved on a combination of three columns arranged in sequence D2000, D2500, D3000 (single-pore - 300 × 8 mm) (Malvern-Panalytical, MA, USA) at 50 °C, with DMF (0.02 M ammonium acetate) as eluent at a flow rate of 0.75 mL/min.

2.5. Poly(anhydride-ester)Gemcitabine Nanoparticle Preparation

Poly(GMT) NPs were prepared using a dialysis-based method.[55,56] The polymer (20 mg) was dissolved in 5 mL of DMF and dialyzed against 500 mL of DI water in a regenerated cellulose dialysis membrane (Spectra/Por) with molecular weight cutoff (MWCO) of 1,000 Da. The water was exchanged every hour during a 3 hours period. The particles were recovered through centrifugation, frozen and lyophilized overnight. The dried particles were stored on inert atmosphere at −4 °C.

2.6. Particle Size, Morphology and ζ Potential

Determination of size and morphology of the polyGMT NPs was performed using a scanning electron microscope (Hitachi SU-70 FE-SEM) at 15 kV. Samples were sputtered with gold before analysis on a Polaron SEM Coating System (Artisan Technology Group, IL, USA). The average geometric diameter was obtained by manually processing (using ImageJ v1.50b) 50 micrographs of NPs. Hydrodynamic diameter (HD), polydispersity index (PDI) and ζ Potential were obtained using a Zetasizer (Malvern-Panalytical, MA, USA). A dispersion of 1 mg/mL was prepared in DI water for all the assays.

2.7. PolyGMT Nanoparticles Release Profile

PolyGMT nanoparticles (20 mg) were dispersed in 2 mL of phosphate buffered saline (PBS) pH 7.4 and added to a dialysis bag (MWCO = 1,500 Da). The bag was, then, immersed in 30 mL of PBS (pH= 7.4). Release studies were performed by gently shaking the system at 37 °C in the absence of light. In determined time points, 0.1 mL of the external solution was collected and analyzed through an adapted HPLC method.[57] Briefly, the analyses were performed using a Zorbax SB-aq (3.5 μm, 2.1× 150 mm) column (Supelco, PA, SA) on an Agilent 1200 system (Agilent, CA, USA) with an UV-Vis detector. The system was connected to a computer running Agilent Chemstation software (B.04.01 SP1). Samples were filtered using 0.22 μm nylon filters (VWR International). The mobile phase was composed of 97% aqueous solution (10 mM dihydrogen phosphate buffer, pH= 3) and 3% acetonitrile. Samples (10 μL) were run at 25 °C at a flow rate of 0.4 mL/min. Absorbance was monitored at λ= 276 nm. The concentration of GMT was determined with respect to an established calibration curve. The experiments were run in completely independent (new batch of nanoparticles and new run) triplicates. After every sample was withdrawn, fresh PBS was added for the maintenance of the initial volume. The cumulative release of GMT from the studied systems was plotted as a function of time.

2.8. Particle/Polymer Accelerated Hydrolysis

In vitro accelerated hydrolysis of the synthesized compounds was performed to assess the recovery of GMT. The drug as received (GMT), GMT-2SA, and polyGMT (20 mg in all cases) were dissolved/dispersed in 2 mL of DI water and added to a dialysis bag (MWCO = 1,500 Da). The bag was, then, immersed in 30 mL of an aqueous 0.1 M NaOH solution (pH= 10). The release was performed by gently shaking the system at room temperature in the absence of light. In determined time points, 0.1 mL of the external solution was collected, neutralized with a 0.1 M HCl solution, and analyzed through the HPLC method described previously. The concentration of GMT was determined with respect to an established calibration curve. The experiments were run in completely independent (new batch of polymer and new run) triplicates. After every sample was withdrawn, fresh NaOH aqueous solution was added for the maintenance of the initial volume. The cumulative release of GMT from the studied systems was plotted as a function of time.

2.9. Cell Culture and Assays

2.9.1. Materials

Cell culture media “Dulbecco’s Modified Eagle’s Medium” (DMEM) 1X high glucose and Trypsin-EDTA (1X) were purchased from Corning (Corning, NY, USA). Antibiotics (Penicillin and Streptomycin) were purchased from Invitrogen (Karlsruhe, Germany). Phosphate buffered saline (PBS, 1X, pH= 7.4) and Hank’s Balanced Salt Solution (HBSS, 1X, pH= 7.4) were prepared in the laboratory. Fetal bovine serum (FBS, nonheat inactivated) was purchased from Atlanta Biologicals (Flowery Branch, GA), tissue culture flasks (T25 and T75) were purchased from VWR International (Radnor, PA, USA). Round bottom 96 wells plates (Cellstar) were purchased from Greiner Bio-One (Kremsmünster, Austria). Flat bottom 96 wells plates were purchased from Corning (Corning, NY, USA).

2.9.2. Passaging and Cell Culture

All cells were passaged in T25 or T75 flasks at 37 °C in 5% CO₂, humidified atmosphere, on HERACELL VIOS 160i incubators (Thermo-Fisher Scientific, Waltham, MA, USA). A549 (human adenocarcinoma alveolar epithelial cells), and K7M2 (mouse osteosarcoma cells) were both purchased from ATCC (Manassas, VA, USA) and tested for mycoplasma prior to use. Cells were grown in DMEM medium, supplemented with 10% FBS, and 1% antibiotic. The cells were, then, shortly washed with PBS solution pH 7.4 and subsequently incubated with fresh trypsin/EDTA solution at 37 °C for 3 minutes. EDTA was removed by centrifugation of the cell suspension at 1,500 rpm for 5 minutes. The cells were, then, resuspended in fresh medium. Cells were counted using a hemocytometer.

2.9.3. A549/K7M2 Cell Viability

A549 or K7M2 cells were seeded in 96 well plates at a density of 10⁴ cells per well. The cells were allowed to grow (DMEM + 10% FBS + 1% AB) for 24 hours at 37 °C and 5% CO₂. Free GMT, GMT-2SA and hydrolysis products of poly(GMT) were added at different GMT equivalent concentrations (GMTeq) between 0.1–1,000 nM. Cell viability with SA, the linker, was also investigated. After administration of the dose, the cells were incubated for an additional 24 hours under 5% CO₂ at 37 °C in DMEM + 10% FBS +1% AB. Wells with no treatment were used as (negative) control. Cell viability was analyzed using MTT Assay (Invitrogen, Thermo-Fisher Scientific, Waltham, MA, USA). Briefly, the cells were rinsed twice with 1X HBSS and 110 μL of 1.09 mM of MTT (in 1X HBSS) was added to each well, followed by the incubation of the plate for 4 h at 37 °C and 5% CO₂. Subsequently, 75 μL of solution was removed from each well and 50 μL of DMSO was added and mixed. The plates were, then, covered and incubated at 37 °C for additional 10 minutes. The absorbance was read at 540 nm using a multi-mode microplate reader (Synergy H1, Biotek, Winooski, VT, USA). At least 6 repeats (n=6) were performed for each condition. Cell viability was calculated using Equation 1.

$$\%CellViability=\frac{UVAbsorbanceoftreatedcells}{UVAbsorbanceofnontreatedcells}x100\%$$ Eqn. (1)

2.9.4. Statistical Analysis and Image Processing

All data are presented as a mean ± standard deviation (s.d.). The means were calculated from a minimum of at least three independent measurements (n ≥ 3). ImageJ (version 1.50b) was used to measure geometric size, and to process micrographs as necessary.

3. Results and Discussion

3.1. Synthesis and Characterization of Monomer/Repeat Unit

The synthesis of a poly(anhydride) chain is the result of the interconnection of two carboxylic acid groups (-COOH), with elimination of a water molecule during the reaction.[43] As the molecule of GMT does not have this necessary functionality, the first synthetic step consisted in reacting the active ingredient with two molecules of 4-(benzyloxy)-4-oxobutanoic acid (SA-Bz), a succinic acid molecule, with one protected -COOH group (Scheme 1).

The use of this linker, in opposition of the direct use of succinic acid (SA), promoted the decrease in the overall polarity of the synthesized molecule and that allowed the purification of the product using normal phase silica-based chromatography. GMT is a very polar compound, bringing several challenges and limitations for chromatographic methods in both the synthetic and analytical fields.[58,59]

Using a mixture of dichloromethane and methanol as eluent, compound 1 (GMT-2SA-Bz) was obtained, with high purity, and its structure was assessed by ¹H NMR. The ¹H NMR spectrum of compound 1 (Figure 1 – II) demonstrated the presence of the functionality, with peaks in the range 2.45–2.64 ppm and at 5.10 and 7.32 ppm, which are related to the -CH₂ groups and aromatic ring of the protective groups, respectively. Comparing the spectrum of GMT-2SA-Bz with the one of GMT (Figure 1 – I), it is possible to observe the change in chemical shift of the hydrogens belonging to carbons atoms connected directly to the -OH groups (peaks 1 and 3) involved in the formation of the ester bond between GMT and the two linker molecules. It is also possible to observe the disappearance of the peaks related to -OH groups (5.17 and 6.21 ppm), after the esterification reaction.

Figure 1.

¹H NMR spectra of gemcitabine (GMT, I), GMT-2SA-Bz (II), and diacid gemcitabine (GMT-2SA, III). (Full assigned spectra available in Support Information)

The resulting GMT-2SA-Bz was, then, submitted to a deprotection step using a hydrogenolysis method.[60,61] A supported catalyst (Pd/C) was used, while the reaction was kept under a H₂ atmosphere. This reaction led to the removal of the benzyl protective groups, resulting in the desired monomer, a diacid modified GMT (compound 2 - GMT-2SA). The ¹H NMR spectrum of this compound demonstrated the disappearance of the peaks related to the protective groups (benzyl) with the maintenance of the peaks related to the (-CH₂)₂ groups from the linker, as those in the range 2.53–2.73 ppm (Figure 1 – III).

All synthesized compounds were also analyzed with MALDI-ToF analysis (Figure 2), where is possible to observe the difference of ~180 Da between the protected species (Figure 2 – A) and the deprotected GMT-2SA (Figure 2 – B), which corresponds to the mass of two benzyl groups.

Figure 2.

MALDI-ToF spectra of GMT-2SA-Bz (A), GMT-2SA (B), and acetylated gemcitabine (GMT-2SA-Acetyl) (C)

One of the most common methods to synthesize poly(anhydride) polymers is the use of a melt condensation reaction,[43,44,62] mainly because of its simplicity, as it uses solely the monomer as reactant, thus avoiding the use of catalysts or any other chemical entities that would need to be removed from the final product.[63] In the present study, the absence of other chemical compounds to promote the reaction is of great importance since the polymer is designed to be a drug delivery system. The presence of other entities could lead to the need of subsequent purification steps as they could, otherwise, bring undesired (toxic) biological effects.

Based on the considerations above, a melt condensation polymerization reaction was used to promote the interconnection of the monomer (GMT-2SA). Even though this type of polymerization could, in principle, be performed directly with the diacid species as it has been shown in literature for other repeat units,[43,64] acetylation of the carboxylic groups has been used as a strategy to improve the characteristics of the final synthesized macromolecule, such as Mw/Mn and PDI.[62] This acetylation step is fundamental to decrease the melting point of the monomer, since diacids usually present very high melting points. The acetylation of the -COOH groups allowed the polymerization reaction to be performed at temperatures below the degradation temperature of the monomer and the growing polymer.

Based on that, the acetylation of GMT-2SA was performed using an excess of acetic anhydride[64,65] and that led to the acetylation of the carboxylic groups (Scheme 1 - compound 3) as well as the formation of dimers, trimers and small oligomers. MALDI-ToF analysis was used to assess the molecular weight of the synthesized molecules. On the final step (synthesis of compound 3), the mass increase of ~43 Da demonstrated the addition of the acetyl group (Figure 2 – C), while the higher molecular mass species observed are multiples of the mass of the diacid GMT-2SA (dimer). It is worth noticing the presence of adducts containing sodium, leading to an additional ~23 Da in the observed masses. After all the described modifications, the derivatized GMT-2SA-Acetyl (compound 3) had the required characteristics to be used in a melt condensation polymerization.

3.2. Synthesis and Characterization of Poly(anhydride-ester)Gemcitabine (polyGMT)

As mentioned previously, a melting condensation reaction was selected as the approach to prepare the polymeric drug (Scheme 2). In this approach, the acetylated monomer (compound 3) was placed in a reactor and submitted to high temperature and low pressure, being the melted reaction mixture stirred with the help of an overhead mechanical stirrer.

Scheme 2.

Scheme of the polymerization of acetylated gemcitabine (GMT-2SA-Acetyl) to poly(gemcitabine) (polyGMT).

The degradation temperature of the designed monomer was taken in consideration when establishing the reaction temperature. TGA results (Supporting Information - Fig S9) showed that the monomer degradation temperature is at 123°C. Th reaction was, then, performed at 120 °C, ensuring the melting of the monomer, and at the same time avoiding its degradation. The reaction was driven by the high temperature applied to the system and elimination, through low pressure, of the residual acetic anhydride/acetic acid formed after the interconnection of the carboxylic acid groups.[44,52] The end point for the reaction was established as when maximum viscosity was reached - viscosity in which our set up could still stir the reaction system, which was ca. 30 min, resulting in polyGMT (compound 4). After the reaction, the product was precipitated in diethyl ether, allowing the removal of any residual acetic anhydride/acid that may not have been removed under low pressure during the reaction. The isolated poly(anhydride-ester) was characterized via ¹³C NMR, FTIR and GPC.

The ¹³C NMR spectra of the modified GMT used as monomer (GMT-2SA) as well as of the resulting polymer (polyGMT) are shown in Figure 3 – II and III. The comparison between the spectra indicates that peaks related to the structure of the monomer (drug + linker) are still present in the final product, showcasing that the chemical structure of the monomer, and ultimately of GMT, was maintained during the polymerization reaction. Due to the chemical environment and abundance, some peaks of the polymer have a very low intensity and were not possible to be identified. Through the polymer spectrum (Figure 3 – III) it is possible to observe the appearance of a peak at 170.2 ppm, which is related to the carbonyl group involved on the formation of the anhydride bond – that peak is absent in the monomer spectrum (Figure 3 – II). The final polymer presents several carbonyl species, since the linker structure has four carbonyl groups that are not equivalent due to their position in the molecule, and the extra group from the acetyl portion. After polymerization, it was possible to observe the carbonyl groups involved in the poly(anhydride) backbone as well as the ones present in the terminal acetyl groups, which resulted in a great number of peaks in the range 170–173 ppm on the polymer ¹³C NMR spectrum.

Figure 3.

¹³C NMR spectra of GMT-2SA-Bz (I), diacid gemcitabine (GMT-2SA, II), and poly(gemcitabine) (polyGMT, III). (Full assigned spectra available in Support Information)

On the ATR-FTIR spectra (Figure 4) it is possible to observe the appearance of a new intense band (1715 cm⁻¹) after the conjugation of SA to the GMT molecule. This band is related to the stretch of the C=O bond from the ester and carboxylic groups present on the formed diacid (GMT-SA) molecule. Analogously, on the spectrum of the resulting polymer (polyGMT), besides the band related to the C=O bond of the ester/COOH groups, it is possible to observe an additional band at 1829 cm⁻¹, which corresponds to the stretching of a C=O bond in the anhydride group.

Figure 4.

ATR-FTIR spectra of Gemcitabine (black), diacid gemcitabine (GMT-2SA, blue) and poly(gemcitabine) (polyGMT, red)

The GPC chromatogram of polyGMT is shown in Figure 5. The traces observed were the ones generated by the refractive index (RI) and light scattering (LS) (other detectors not shown). The delimited area was selected based on the total elution of the macromolecules and was used for the determination of the molecular weight of the polymer.

Figure 5.

GPC analysis of poly(gemcitabine) (polyGMT). Dashed lines delimit the region used for calculation of average masses (Mw/Mn). DMF was used as eluent.

The GPC setup has tetra detection, which allowed the determination of all characterization parameters without the need for column calibration.[66] All the parameters were processed through OmniSEC software (version 10) and are summarized in Table 1.

Table 1.

Characterization of polyGMT

Mw (Da)	Mn (Da)	PDI	Rh (nm)
19,740	12,210	1.6	1.4

The values obtained for Mw, Mn and polydispersity index (PDI) demonstrated consistency, and are in agreement with similar polyanhydrides synthesized with bio-active ingredients.[40,50–52]As an example, polymorphine, also a poly(ester anhydride), has a Mw=26,100 Da.[51] The reproducibility of the method was evaluated as well and the results, in terms of Mw and Mn, of four totally independent batches resulted in a relative standard deviation of 12%.

Based on the results obtained, and the fact that each polymeric chain is composed of ca. 26 molecules of GMT, the drug load was determined to be 58%w/w, which is the highest of any previously published on encapsulated/conjugated GMT drug delivery system.[67,68] This high drug loading implies also that the ratio of inactive/active ingredients in this polymeric drug delivery system is very low, being the inactive species limited to a small molecule (SA - Mw: 118 Da) with very high biological compatibility.[69] As comparison, traditional nanoparticle systems used to formulate GMT, where the drug is encapsulated inside the particle composed of an inactive polymer (eg. PLGA), [34,35] or liposomal formulations,[70,71] usually have drug loadings of < 5%w/w.

Another important polymer characteristic for drug delivery applications is the thermal properties of the synthesized polyGMT. The thermogram of the polymer (Figure 6) shows that the polymer has a high glass transition (Tg) temperature at 123 °C. This value is in the same range for other poly(anhydrides) and poly(anhydrides-esters), with comparable backbone structures.[72]

Figure 6.

DSC analysis of poly(gemcitabine) (poly GMT). Inset: Region used for Tg calculation.

As thermal properties are related to the flexibility of the polymeric chain,[73] an significant contribution for a high Tg value of this class of polymers is the fact that the they have a rigid anhydride bond as the connection between the monomers.[74] In the present case, another factor that contributed for the observed Tg value is the group selected to derivatize GMT with, namely SA. The overall flexibility of the chain is impacted by the short hydrocarbon chain linker as well (-CH₂)₂. Other polymers, of same nature, but containing longer hydrocarbon linkers or other types of chemical bonds (eg. ether) demonstrate lower Tg values, likely due to the impact on the overall chain flexibility.[50,65,75]

The value of Tg for polyGMT is of great relevance for the use of this polymer in the development of drug delivery systems. Values of Tg higher than room temperature (25 °C) allows the polymer to be stably stored at ambient conditions after being engineered as nanoparticles.[72,76] A Tg value higher than the physiological temperature (37 °C) also results in the stability of the engineered systems upon contact with the biological environment (eg. i.v. administration), keeping the desired 3D structure upon use.[77]

3.3. Engineering Nanoparticles (NP) of polyGMT

There are several potential constructs that can be envisioned for such polymers, from macroscopic implants[49] to nanostructured drug delivery systems.[77] In this work we focus on demonstrating feasibility of preparing NPs with polyGMT given the relevance of the active compound in chemotherapies, and the significant potential clinical benefit of polymeric NPs in cancer treatment, as indicated by the large number of ongoing clinical trials.[36,37,78–80] The relevance of NP-based delivery has been demonstrated by their ability to enhance the pharmacological properties of drugs, including in vivo stability, pharmacokinetic profile, and ability to protect the drug from a premature and off-site release.[81] Additionally, NPs have demonstrated differentiated interaction with the tumor microenvironment, potentially allowing a more efficient penetration of the drug carrier.[82,83] As free GMT has poor chemical stability under physiological conditions and poor biodistribution / tumor site accumulation,[11] establishing the feasibility of engineering NP with the synthesized PolyGMT is a relevant next step towards a more efficient drug delivery system for safe and effective use of this broadly applicable chemotherapeutic.

The synthesized polyGMT demonstrated a very limited solubility in a variety of solvents, being highly soluble in selected polar solvent, including DMF and DMSO.[84] This characteristic limited the feasibility of several approaches for the engineering of the polymer into particles.[85] One of the possible procedures to circumvent these limitations was the use of a dialysis-based method.[55,56] In this approach, polyGMT was first solubilize in DMF (good solvent) and this solvent was exchanged by water (poor solvent), having a dialysis membrane to restrict the movement of the formed nuclei/growing particles upon solvent exchange. In this process, the MWCO of the dialysis membrane plays an important role on the final particle size.[56] In the present study, a MWCO membrane with 1,500 Da cut off diameter was used. The selection of the MWCO of the membrane was based on the Mw of the synthesized polymer, in an attempt to mitigate losses during the process.[56] Due to the poor solubility in water, during the increase of water fraction inside the dialysis bag, the polymer crashes out of the solution nucleating small particles that grow to become an aqueous dispersions. The controlled solvent exchange led to the preparation of NPs with dimensions in the nanoscale. The shape and size of the particles were assessed through SEM (Figure 7).

Figure 7.

SEM micrographs of poly(gemcitabine) (polyGMT) nanoparticles produced through a dialysis-based method.

As can be observed from the micrograph, the NPs have uniform shape and size, demonstrating the efficiency of the approach used. Additionally, as presented in the Table 2, the particles were characterized regarding their hydrodynamic diameter (HD) and zeta potential (ζ) (Supporting Information - S10–S11 for details).

Table 2.

Characterization of polyGMT Nanoparticles

DLS		SEM	ζ (mV)
HD (nm)	PDI	Diameter (nm)
229 ± 28	0.46 ± 0.05	206 ± 38	−12.0 ± 1.4

The engineered particles demonstrated a size compatible to the range established as ideal for improving the formulation performance in terms of PK and biodistribution (~200 nm).[86] The negative ζ potential is high enough to maintain particles well dispersed in DI water and PBS.[87] The particles demonstrated stability in aqueous medium, keeping their HD over 10 days, as assessed by DLS.

In summary, this new biomaterial synthesized here has thermophysical properties suitable for the engineering of NPs, which are stable at ambient and physiological conditions and also have appropriate size and zeta potential to be used as a drug delivery system.

3.4. In vitro controlled release of polyGMT Nanoparticles

Polyanhydrides are a class of polymers with unique properties in terms of their hydrolysis profile.[43,74,88–90] This class of polymer, when engineered in a 3D structure, undergoes a surface erosion process, where only the external and exposed surface undergoes hydrolysis.[89] The hydrolysis of polyanhydrides thus follow a zero order profile, which suits this class of material as drug delivery systems.[62] Because, in this work, the drug is part of the polymeric chain, the drug release follows the hydrolysis rate of the engineered NP and polymer, thus avoiding any initial burst release, and being independent of the behavior of any carrier polymer as would be the case for free GMT encapsulated within inactive polymer NPs.[91] Different from common polyanhydrides, however, polyGMT, has two hydrolytic processes that need to occur before GMT is retrieved as free drug. In the first step, the water exposed polymer on the surface of the NPs would hydrolyze the anhydride bond, releasing the pro-drug (GMT-2SA), which, in turn, will suffer a second hydrolytic process to release the SA linkers and the free GMT.

The release of GMT from polyGMT NPs in PBS (pH= 7.4; 37 °C) is shown in Figure 8. One can observe the ultra-long release kinetics of these NPs. Along 45 days, the cumulative release of GMT was 33% of the total amount, having the process, during the period of time studied, followed a close to zero-order kinetics. The rate of release was calculated as 0.7% per day. This behavior is similar to that observed on other polyanhydrides using (CH₂)₂ groups as linkers, where the use of such small and hydrophobic groups promotes a reduction in the rate of release.[75] The hydrolysis of polyanhydrides with this specific (or equivalent) linker has demonstrated that less than 10% of the engineered material (in this case a disk) is hydrolyzed within 10 days.[75,77] Such slow release profile polymers have been explored in the fields of implants[46] and injectable drug delivery systems.[49]

Figure 8.

Cumulative release of poly(gemcitabine) (polyGMT) nanoparticles on buffered aqueous solution (pH 7.4) at 37 °C as a function of time (n=3).

Such ultra-long drug release profile systems find potential applications as implants in hard to reach (poor bioavailability) tumors, as for example high grade malignant glioma, similarly to Gliadel®, a chemotherapy infused polyanhydride wafer currently in clinical use.[92] It is also worth pointing out that the proposed approach is a platform technology that allows us to explore various linker chemistries in order to fine tune the release kinetics of the chemotherapy for other applications such as those tumors that can be targeted upon systemic drug administration.

3.5. In vitro Release of polyGMT Nanoparticles via Accelerated Hydrolysis

To demonstrate the ability to fully recover GMT upon complete hydrolysis of the NPs, and maintenance of the biological activity of the active ingredient (GMT) after the synthetic (monomer preparation and polymer synthesis) and hydrolytic processes, polyGMT NPs were also subjected to an accelerated hydrolysis process by exposing them to basic conditions (pH 10). This alkaline media was not intended to mimic any physiological condition, but instead, it was selected due to the fact that anhydride and esters bonds have demonstrated faster hydrolysis under these conditions.[93] The accelerated release was followed by determining the concentration of free GMT with HPLC as a function of time. The results are shown in Figure 9.

Figure 9.

Cumulative release of gemcitabine (GMT) after accelerated hydrolysis (0.1 M NaOH - pH 10) during 6 hours (A) GMT as received; (B) diacid gemcitabine (GMT-2SA), and (C) poly(gemcitabine) (polyGMT). Insets: Cumulative release for 48 h. (n=3).

The initial 6h of the hydrolysis process is shown in Figure 9. The experiment followed exposures for up to 48h to evaluate the stability of the free drug (insets - Figure 9). The hydrolysis of the monomer (GMT-2SA) was also studied. As control, free drug (GMT) was submitted to the same conditions. This control experiment served two purposes; one was to evaluate the losses inherent to the experiment design, and the second was to access the stability of the free drug under those alkaline conditions.

The control experiment (Figure 9 – A) shows that 3% of the drug is lost due to experimental limitations, and this loss is very similar to that seen for the monomer and polyGMT NPs. Additionally, the results demonstrate that that free GMT is stable under such conditions during the length of the experiment (48 h). Both the monomer (GMT-2SA) (Figure 9 – B) and the polyGMT NP (Figure 9 – C) demonstrated similar release profiles to the control experiment. After 1 h, the hydrolysis process was completed, and the release reached the maximum value in all cases, 98% for GMT-2SA and 97% for polyGMT. In this experiment, the high recovery of free GMT after exposure to basic conditions indicates that hydrolysis of both anhydride and ester bonds were successful. The accelerated hydrolysis experiment provided a way to recover the totality of the GMT from the polymer chains in a reasonable amount of time, and thus, allowed subsequent comparative studies regarding efficacy.

3.6. Biological Activity of Released GMT: In Vitro Cytotoxicity to Tumor cells

GMT is used in the treatment of several types of cancers, alone or in combination with other drugs.[11,94] To demonstrated that GMT released from polyGMT still keeps its biological activity after hydrolysis, the cytotoxicity of the depolymerization products was evaluated in A549 human alveolar epithelial cells, and also in K7M2 cells, a relevant model of immunocompetent murine osteosarcoma. The assay was performed with the products of hydrolysis, because the ultra-long release time scales of GMT from the polyGMT NPs is much greater than typical time scales for in vitro assays. These results are summarized in Figure 10.

Figure 10.

Viability of: (A) A549 cells and (B) K7M2 cells, determined by the MTT assay after 72 h incubation, after treatment with: gemcitabine (GMT), diacid gemcitabine (GMT-2SA) and hydrolysis products of poly(gemcitabine) (polyGMT). Results denote mean ± s.d. (n=6).

As can be seen, the profiles obtained with the products of hydrolysis of polyGMT are very similar to those for the free drug, demonstrating that the drug biological activity is largely maintained after all the synthetic steps used for the polymer formation and after the hydrolysis processes. The IC₅₀ for the free drug in A549 was calculated as 4 nM, while that for GMT after hydrolysis of polyGMT NPs, that include SA as a by-product, is 7 nM. In comparison, the value for IC₅₀ for free GMT in K7M2 was 18 nM, while that for the products of hydrolysis of polyGMT was 28 nM.

As expected, SA does not lead to further toxicity (IC₅₀ does not decrease in the presence of released SA along with GMT), and small discrepancies may arise due differences between estimated and actual loading of GMT, as the total concentration of GTM in the hydrolyzed medium was based on an average Mw and average number of GMT (due to polymer Mw/Mn distribution). The values reported for A549 are in agreement with those for free GMT observed in the literature.[95,96] It is worth mentioning that due to fact that GMT has both agonist and antagonist effects on A459 cells,[97] it is possible to observe two inflections points and calculate two values for IC₅₀. We limited our discussion to the first inflection point, which is typically reported in the literature.

Finally, the cytotoxicity of the monomer (GMT-2SA) was also evaluated in both cell lines. The results demonstrated that the hydrolysis and elimination of the SA groups is a fundamental step for the recovery of the biological active GMT. That could be related to the fact that the -OH group in the position 5’ needs to be phosphorylated upon cell entry of GMT, as a first step to produce the active form of the drug.[94]

In this assay we demonstrated that GMT keeps its biological activity after the synthesis and hydrolysis of the polymer. We are currently evaluating the anti-tumor efficacy of polyGMT nanoparticles in vivo.

4. Conclusion

In this work we report the preparation and evaluation of the first poly(anhydride-ester) biomaterial using a chemotherapeutic drug as building block. PolyGMT was synthesized via melt-condensation polymerization and its physicochemical properties were fully characterized to confirm the preservation of GMT’s structural integrity. The obtained material demonstrated a very high payload, being each polymer chain composed of an average of 26 GMT molecules, a payload of 58%w/w. The thermal characterization of the polymer demonstrated the potential of the material to be processed, and stably stored and exposed to the biological environment. NPs of polyGMT were successfully engineered using the dialysis-based method, with resulting NPs having an average diameter of 206 ± 38 nm. The in vitro feasibility release assay demonstrated that the polymer can undergo hydrolysis, with both anhydride and ester bonds being broken in aqueous environment. This process resulted in the total release of the cargo (free GMT) and the total disappearance of the polymeric chain. After hydrolysis, it was demonstrated that the released drug kept its biological activity, as assessed by MTT assays on A459 and K7M2 cells.

The designed drug delivery system has the potential to overcome some of the challenges in the treatment of cancer using GMT, by combining a high payload polymer with its engineering into nanoparticle form. The polymeric structure can act by protecting the cargo from biological deactivation and the use of a nanosized polymeric structures can act by altering the drug pharmacokinetic profile. This new technology can be use as platform to prepare polyGMTs with varying characteristics to suit needs by changing the linker chemistry, and also to prepare other polymer chemotherapeutics by modifying functional groups that will allow us to recover the active compound.

6. Data Availability

The raw data required to reproduce these findings are available to download from https://osf.io/tpdcq/?view_only=68c31de407084814a47596289b4552e3. The processed data required to reproduce these findings are available to download from https://osf.io/tpdcq/?view_only=68c31de407084814a47596289b4552e3.