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. Author manuscript; available in PMC: 2026 Feb 24.
Published in final edited form as: J Appl Polym Sci. 2025 Feb 24;142(21):e56928. doi: 10.1002/app.56928

Synthesis and Characterization of Polymer-Drug Conjugates by Strain-Promoted Azide-Alkyne Cycloaddition-Mediated Polymerization

Omotola D Gbadegesin 1, Simeon K Adesina 1,*
PMCID: PMC12233213  NIHMSID: NIHMS2060965  PMID: 40630867

Abstract

Polymer-drug conjugates (PDCs) modify the biodistribution of small-molecule anticancer agents to prevent undesired off-target adverse effects. Here, we report the preparation of two PDCs by strain-promoted [3 + 2] azide-alkyne cycloaddition-mediated step-growth polymerization. This method does not require the use of a catalyst or high temperatures and it allows the rapid synthesis of high molecular-weight PDCs containing gemcitabine (Mw ~ 40.18 kDa) and doxorubicin (Mw ~ 1800 kDa) with narrow molecular weight distribution and high drug loading (29.2 %wt. gemcitabine and 10.3 %wt. doxorubicin). α-ω-bis-azide-terminated bifunctional gemcitabine-coupled and doxorubicin-coupled monomers, with drug linkage via Gly-Phe-Leu-Gly (GFLG), a cathepsin B-sensitive peptide linker, were separately synthesized and polymerized using a dibenzoazacyclooctyne bifunctional polyethylene glycol monomer. Preliminary in vitro evaluations of the PDCs showed cathepsin B-catalyzed drug release at pH 5.0. The applied method for the syntheses of the PDCs enables the selective delivery of potent anticancer agents.

Graphical Abstract

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1. Introduction

A major problem of traditional cancer chemotherapy is the lack of selective toxicity resulting in unwanted systemic toxicity and serious adverse effects.1,2 In addition to killing cancer cells, chemotherapeutic agents also damage healthy cells, limiting the maximum dose that can be safely administered to an individual patient.1,3 Drug delivery approaches that have the potential to improve the efficacy and decrease the toxicity of current chemotherapeutic drugs are, therefore, required. One such approach is the delivery of chemotherapeutic agents as polymer-drug conjugates (PDCs). PDCs are drug delivery systems where active pharmaceutical agents are covalently attached to polymeric chains through stimuli-sensitive linkers.4–6

PDCs offer opportunities such as high drug loading7 and modification of the pharmacokinetics of the conjugated drugs to prevent unwanted distribution to healthy tissues.3 Based on their macromolecular size, PDCs can preferentially accumulate in cancer tissues as a result of the tumor’s leaky vasculature and impaired lymphatic drainage via the enhanced permeability and retention (EPR) effect.5,8 This is referred to as passive targeting. Additionally, the site-specific expression or overexpression of certain enzymes in cancers with negligible or no expression in healthy tissues increases the targeting specificity of PDCs. A common example of such enzymes is cathepsin B, a lysosomal enzyme that is highly overexpressed and secreted in many solid tumors.9,10 Different types of cathepsins, such as cathepsin A, B, C, F, D, and L, perform many functions such as proteolytic degradation of cellular proteins, intracellular housekeeping, and apoptotic signal transduction, in cells.11,12 Specific cathepsins (such as cathepsins B and L) are often upregulated in various cancers and have been implicated in cancer progression and metastasis.9,12 Dipeptides (such as valine-citrulline and valine-alanine) and tetrapeptides (such as glycine-phenylalanine-leucine-glycine and glycine-glycine-leucine-glycine have served as cathepsin B-cleavable linkers in many cancer-targeted drug conjugate systems.6,13,14

In this study, glycine-phenylalanine-leucine-glycine (GFLG) was selected over cathepsin B dipeptide substrates because the use of oligopeptide linkers minimizes steric hindrance effects that may obstruct the formation of enzyme-substrate complexes.15 In addition, valine-citrulline, which is very popular in the development of commercialized tumor-targeted drug conjugates like antibody-drug conjugates,16 is reported to be sensitive to a variety of cathepsins and could induce non-specific drug release causing off-target toxicity in normal cells.17 GFLG, on the other hand, is more specific for cathepsin B and is stable in the plasma.18–20 It has been reported that the use of GFLG as a cathepsin B substrate linker has some drawbacks, such as hydrophobicity and long cleavage times, which might cause a slower drug release and a consequent reduction in cytotoxic efficacy.21 The idea of cathepsin B-sensitive tetrapeptide linkers was recently validated with the approval of Enhertu™, an antibody-drug conjugate containing deruxtecan (topoisomerase I inhibitor) bound to trastuzumab (a monoclonal antibody) through glycine-glycine-leucine-glycine (GGLG) tetrapeptide spacer, by the U.S. Food and Drug Administration (FDA).15 The selective cleavage of GGLG by cathepsin B will trigger the tumor-specific release of deruxtecan, minimizing toxicity to healthy cells.

Several studies have been published that report the development of PDCs for cancer targeting and treatment. Poly (N-(2-hydroxypropyl) methacrylamide) (HPMA), a linear water-soluble synthetic polymer has been most commonly used for the synthesis of PDCs because of its biocompatibility, non-immunogenicity, and relative ease of incorporating one or more drug molecules and targeting agents.3,7,15 Some HPMA-based PDCs (such as PCNU166148™, PNU166945™, PK1, and PK2) have been clinically tested for the treatment of different cancers.4 CRLX101, a self-assembled cyclodextrin-based nanoparticle-drug conjugate of camptothecin has also been reported.6 In a clinical study involving 29 patients with relapsed platinum-resistant ovarian cancer, CRLX101 showed a clinical benefit rate of 68%.22 The polymeric nature of the conjugate allowed it to accumulate preferentially in tumor tissues, exhibiting sustained slow release of camptothecin while limiting toxicity in healthy cells.23 Despite these efforts, no polymer-drug conjugate is yet approved for cancer treatment.4,5 Major factors that limit the clinical translation of PDCs include polymer safety and toxicity,3,4 short plasma circulation, structure complexity, and batch-to-batch synthetic reproducibility.24 Ideal PDCs should have high drug loading, circulate in the blood to sufficiently accumulate in the tumor tissue by the EPR effect, be internalized by the tumor cells, and efficiently release the drug. Also, the polymer carrier must be fully eliminated from the body after drug release.6 There is, therefore, a growing interest in the development of PDCs based on different polymer backbones in addition to the few existing ones.5

Here, we report the synthesis of two cathepsin B-sensitive PDCs each containing gemcitabine and doxorubicin, respectively, using strain-promoted [3+2] azide-alkyne cycloaddition (SPAAC)-mediated step-growth polymerization. This method is simpler and faster than other methods that have been reported in the literature for the synthesis of PDCs used for the delivery of anti-cancer drugs. For instance, polymer precursors have been copolymerized with polymerizable gemcitabine or doxorubicin derivatives using the reversible addition-fragmentation chain transfer (RAFT) polymerization method.3,25–28 This method has the advantage of forming high molecular-weight PDCs rapidly but also produces initiator species that must be efficiently removed from the reaction system.29 In addition, the reaction conditions commonly employed when using this method involve rigorous controls, many steps of functional group protection and deprotection, and the use of high temperature, which may not be suitable for a lot of monomers, functional groups, or drugs, and may also affect the reaction efficiency.30

Other studies have also conjugated doxorubicin to pre-formed polymers prepared by RAFT19, atom transfer radical31, free radical32, and living cationic ring-opening33 polymerization mechanisms. In addition to requiring rigorously controlled reaction conditions and multiple complex steps, this synthesis method resulted in PDCs with less than 10% weight doxorubicin content. An ideal polymerization process would involve the absence of an initiator or catalyst, rapid progression under mild conditions, tolerance to air and water, absence of any by-products, and easy isolation of the polymerized product.34 To achieve this in the synthesis of drug-coupled polymer conjugates, a lot of focus is now on the use of click-chemistry, which offers the advantage of easy and fast synthesis of polymers with more precise control over the architecture and functional groups.30,34–37

Click chemistry refers to certain reactions that are characterized by their rapidity, versatility, high product yields, high specificity, easy synthesis, and easy isolation of the synthesized product.38,39 Sharpless et al. established criteria for a click reaction, which encompass “modularity, mild reaction conditions, and quantitative yields”.37 There are typically no byproducts produced by most click reactions, which involve carbon-heteroatom bond (mostly N, O, and S) formation.40 Click chemistry therefore offers a simple and high-yielding synthetic route for the synthesis of polymeric systems with different architectures.37

SPAAC-mediated polymerization enables an easy and rapid synthesis of polymers with more precise control over the architecture and functional groups under mild conditions and without a catalyst or initiator.34,35 Polymers with molecular weights greater than 700 kDa have been synthesized by SPAAC-mediated step-growth polymerization reaction between sym-dibenzo-1,5-cyclooctadiene-3,7-diyne and bis-azide monomers.41 The rapid synthesis of polymers with molecular weights up to 89.5 kDa by step-growth polymerization using copper-free SPAAC reaction between bis-DBCO and bis-azide homo-bifunctional monomers has also been reported.34,35 SPAAC is a highly versatile copper-free click reaction that takes place as a result of a fast, spontaneous, and highly specific reaction between azides and cycloalkynes such as bicyclononyne, dibenzocyclooctyne, and dibenzoazacyclooctyne.42 It is also amenable to a variety of functionalized alkyne- and azide-based monomers.30

In this report, α-ω-bis-azide-terminated bifunctional gemcitabine-coupled monomer and α-ω-bis-azide-terminated bifunctional doxorubicin-coupled monomer were separately polymerized with homo-bifunctional DBCO-PEG6-DBCO monomer to generate high molecular weight PDCs of gemcitabine and doxorubicin, respectively, via copper-free click chemistry. The PDCs also demonstrated cathepsin B-catalyzed release of the drugs in vitro.

2. Materials and Method

2.1. Materials

Dimethyl sulfoxide (DMSO), N, N′-diisopropylethylamine (DIPEA), acetonitrile, trifluoroacetic acid (TFA), dichloromethane (DCM), methanol, N, N′-dimethylformamide (DMF), ethylenediaminetetraacetic acid (EDTA) dianhydride, 4-ethyl morpholine, isobutyl 1,2-dihydro-2-isobutoxy-1-quinolinecarboxylate (IIDQ), lithium chloride (LiCl), sodium phosphate monobasic monohydrate, EDTA disodium salt dihydrate, sodium hydroxide pellets, and 1,4-dithiothreitol (DTT) were purchased from Sigma-Aldrich (Burlington, MA, USA). Piperidine, trifluoro acetic acid (TFA), Fmoc-L-leucine, Fmoc-L-phenylalanine, Fmoc-L-glycine, and hexafluorophosphate azabenzotriazole tetramethyl uronium (HATU) were purchased from Chem-Impex International (Wood Dale, IL, USA). Azido-PEG5-amine was purchased from BroadPharm (San Diego, CA, USA). Glycyl-2-chloro-trityl resin was purchased from AAPPTEC (Louisville, KY, USA). Dibenzoazacyclooctyne-hexa (ethylene glycol)-dibenzoazacyclooctyne (DBCO-PEG6-DBCO) was purchased from AxisPharm (San Diego, CA, USA). Purified native human cathepsin B was purchased from Athens Research and Technology (Athens, GA, USA). Gemcitabine hydrochloride and doxorubicin hydrochloride were purchased from Biosynth Limited (United Kingdom). All materials were used as received.

2.2. Synthesis of α-ω-bis-azide-terminated bifunctional drug-coupled monomers

α-ω-bis-azide-terminated bifunctional drug-coupled monomers containing gemcitabine or doxorubicin were synthesized via amide bond formation reactions between carboxylic acid and amino groups. Amide bonds are commonly used in the synthesis of chemical molecules because of their great stability.43,44 These reactions often require the activation of the carboxylic acid either through the use of chemically reactive moieties like acid anhydrides or ester or by adding a coupling agent with the ability to activate the carboxylic acid in situ.43 The synthesis of the two α-ω-bis-azide-terminated bifunctional drug-coupled monomers was carried out in three steps:

  1. Synthesis of (azido-PEG5)2-EDTA acid (1)

  2. Synthesis of GFLG-bearing polymer backbone intermediate, (azidoPEG5)2-EDTA-(GFLG)2 (2) by solid phase peptide synthesis (SPPS) method

  3. Synthesis of α-ω-bis-azide-terminated bifunctional gemcitabine-coupled monomer, (azidoPEG5)2-EDTA-(GFLG-Gem)2 (3), or

  4. Synthesis of α-ω-bis-azide-terminated bifunctional doxorubicin-coupled monomer, (azidoPEG5)2-EDTA-(GFLG-Dox)2 (4)

2.2.1. Synthesis of (azido-PEG5)2-EDTA acid (1)

(Azido-PEG5)2-EDTA acid (1) is a α-ω-bis-azide-terminated, linear hydrophilic derivative of ethylenediaminetetraacetic acid bearing two carboxylic acid groups. It was synthesized by a single-step amide bond formation between azido-PEG5-amine and EDTA dianhydride. Briefly, azido-PEG5-amine (6.2218 g, 0.02030 mol) was dissolved in anhydrous DMSO in a round-bottomed flask. DIPEA (5.4 mL, 0.03124 mol) was added, and EDTA dianhydride (2 g, 0.00781 mol) was added. The reaction was stirred for three hours at room temperature. The resultant solution was concentrated by rotary evaporation (Rotavapor R-300, Buchi, Switzerland) at 37 °C and purified by preparative high-performance liquid chromatography (HPLC). Pure HPLC fractions were concentrated by rotary evaporation followed by drying over sodium sulfate to isolate the desired compound as a light-cream-colored viscous liquid (4.5 g, 66.3%). Analytical HPLC: single peak at retention time 5.036 min (Supporting information, Figure S1); Fourier-transform infrared (FT-IR) spectroscopy: azide asymmetric stretching frequency at 2103.45 cm−1 (Supporting information, Figure S2); proton nuclear magnetic resonance (1H NMR) spectroscopy, 400 MHz, DMSO-d6, δ (ppm): 8.2 – 8.4 (carboxylic acid -OH), 7.7 – 7.9 (secondary -NH), 3.1 – 3.9 (m, repeating -OCH2), and 1.1 – 1.4 (-CH2N3) (Supporting information, Figure S3); electrospray ionization mass spectrometry (ESI-MS): m/z calculated for C34H65N10O16 [M + H]+ = 869.5, found 869.7 (Supporting information, Figure S4).

2.2.2. Synthesis of GFLG-bearing polymer backbone intermediate

(AzidoPEG5)2-EDTA-(GFLG)2 (2) was synthesized by the solid-phase peptide synthesis (SPPS) method.45 The cathepsin B-cleavable tetrapeptide linker, GFLG, with a free terminal amino group, was synthesized on glycyl-2-chlorotrityl resin (12.5 g, 0.787 mmol/g resin substitution) using 9-fluorenyl-methoxycarbonyl (Fmoc)-solid phase peptide synthesis. (AzidoPEG5)2-EDTA acid (1) was reacted with an excess of the resin-bound GFLG (0.4: 1) to form the desired compound. Resin cleavage followed by precipitation into cold ether was done, and the compound was purified by preparative HPLC to obtain the pure compound 2 as a light-yellow colored viscous semi-solid (1.62 g, 26.8%). FT-IR: azide asymmetric stretching frequency at 2106.98 cm−1 (Supporting information, Figure S5); 1H NMR, 400 MHz, DMSO-d6, δ (ppm): 0.83 – 0.89 (dd, terminal -CH3, Leu), 1.1 – 1.35 (-CH2, Leu), 3.4 – 3.9 (m, repeating -OCH2, (azido-PEG5)2-EDTA acid), 7.14 – 7.24 (aromatic -CH, Phe), 8.1 – 8.3 (secondary amide -NH, Leu and Phe), and 8.25 – 8.6 (secondary amide -NH, Gly) (Supporting information, Figure S6); ESI-MS: m/z calculated for C72H118N18O24 [M + H]+ = 1618.9, found 1618.2 (Supporting information, Figure S7).

2.2.3. Synthesis of α-ω-bis-azide-terminated bifunctional gemcitabine-coupled monomer

Gemcitabine was coupled to (azidoPEG5)2-EDTA-(GFLG)2 (2) using HATU to obtain (azidoPEG5)2-EDTA-(GFLG-Gem)2 (3), an azide-terminated homo-bifunctional gemcitabine-coupled monomer. To liberate gemcitabine base from its hydrochloride salt, DIPEA (0.86 mL, 4.9141 mmol) was added to gemcitabine HCl (0.73628 g, 2.4571 mmol) before adding DMF as solvent. (AzidoPEG5)2-EDTA-(GFLG)2 (2) (0.99377 g, 0.61426 mmol) was dissolved in anhydrous DMF and added to the clear solution of gemcitabine, followed by the addition of HATU solution in DMF (0.70068 g, 1.84279 mmol). The reaction was monitored with mass spectrometry. After 165 min, the solvent was removed by rotary evaporation and the product was purified by preparative HPLC. The pure fraction was lyophilized to obtain pure compound 3 as a white solid (0.4569 g, 35.3%). Analytical HPLC: single peak at retention time 6.640 min (Supporting information, Figure S8); FT-IR: azide asymmetric stretching frequency at 2101.26 cm−1 (Supporting information, Figure S9); 1H NMR, 400 MHz, DMSO-d6, δ (ppm): 0.83 – 0.89 (dd, terminal -CH3, Leu), 1.1 – 1.35 (-CH2, Leu), 3.4 – 3.9 (m, repeating -OCH2, (azidoPEG5)2-EDTA-(GFLG)2), 6.15 – 6.25 (t, -CH, gemcitabine furan ring), 6.3 – 6.4 (d, -CH, gemcitabine pyrimidine ring), 7.14 – 7.24 (aromatic -CH, Phe), and 11.05 (d, -CH, gemcitabine pyrimidine ring) (Supporting information, Figure S10); ESI-MS: m/z calculated for C90H137F4N24O30 [M + H]+ = 2110.0, found 2109.3 and 1055.2 [M + 2H]2+ (Supporting information, Figure S11).

2.2.4. Synthesis of α-ω-bis-azide-terminated bifunctional doxorubicin-coupled monomer

The second drug-coupled monomer, (azidoPEG5)2-EDTA-(GFLG-Dox)2 (4), was synthesized by coupling doxorubicin to (azidoPEG5)2-EDTA-(GFLG)2 (2) using IIDQ.45 To prevent the rapid degradation of doxorubicin in the presence of a strong base, morpholine (less basic than DIPEA) was used to liberate doxorubicin from its hydrochloride salt using the method of Van Heeswijk et al.46 with slight modification. Briefly, compound 2 (200 mg, 0.12362 mmol) was dissolved in DMF. In a separate vial, 4-ethyl morpholine (62.58 μL, 0.49449 mmol) was added to doxorubicin HCl in DMF (0.2868 g, 0.49449 mmol), and transferred into the reaction vial containing compound 2. After stirring for 1h, IIDQ solution in DMF (0.188 g, 0.618115 mmol) was added to the reaction, and the reaction was left to continue stirring in the dark for 42 h and purified by preparative HPLC. The pure fraction was concentrated by rotary evaporation, lyophilized, and obtained as a bright-red solid (0.109 g, 33.1%). Analytical HPLC: single peak at retention time 8.164 min (Supporting information, Figure S12); FT-IR (azide asymmetric stretching frequency at 2101.26 cm−1) (Supporting information, Figure S13); 1H NMR, 400 MHz, DMSO-d6, δ (ppm): 0.75 – 0.81 (dd, terminal -CH3, Leu), 1.11 – 1.13 (dd, -CH3, doxorubicin amino sugar), 3.2 – 3.7 (repeating -OCH2, (azidoPEG5)2-EDTA-(GFLG)2), 4.6 (O-CH3, doxorubicin), 7.15 – 7.25 (aromatic -CH, Phe), 7.45 – 7.9, 7.8 – 7.9 (aromatic -CH, doxorubicin), 13.23 and 13.99 (aromatic -OH, doxorubicin) (Supporting information, Figure S14); ESI-MS: m/z calculated for C126H173N20O44 [M + H+] = 2670.2, found 2670.6 (Supporting information, Figure S15).

General Purification and Characterization Techniques

The synthesized compounds (1 – 4) were purified by preparative HPLC (Agilent 1290 Infinity II Preparative LC System, Agilent Technologies, Santa Clara, CA; solvent gradient: 10% v/v acetonitrile increased to 90% v/v acetonitrile; flow rate: 50 mL/min; detection wavelength: 280 nm; run time: 18 minutes. The mobile phase contained 0.1% v/v TFA). They were characterized by analytical HPLC (Agilent 1100 Series LC, Agilent Technologies, Santa Clara, CA), FT-IR spectroscopy (Perkin Elmer Spectrum 100 FT-IR spectrometer, Perkin Elmer, Waltham, MA), 1H NMR spectroscopy (Bruker AVANCE 400 MHz NMR spectrophotometer, Bruker, Billerica, MA), and positive mode electrospray ionization mass spectrometry (Agilent Accurate-Mass TOF LC/MS, Agilent Technologies, Santa Clara, CA).

2.3. Synthesis of gemcitabine and doxorubicin PDCs by SPAAC-mediated step-growth polymerization

For the synthesis of gemcitabine-coupled polymer conjugate (p-Gem), solutions of (azidoPEG5)2-EDTA-(GFLG-Gem)2 (3) (0.03224 g; 38.2 mM in 0.4 mL of methanol) and DBCO-PEG6-DBCO (0.01375 g; 38.2 mM in 0.4 mL of DCM) were separately prepared. DBCO-PEG6-DBCO solution (0.7 equivalent; 0.28 mL) was added to the solution of (azidoPEG5)2-EDTA-(GFLG-Gem)2 (3) and vortexed before removing the solvent mixture by rotary evaporation (37 °C, 60 rpm). The remaining 0.3 equivalent of the DBCO-PEG6-DBCO solution was added in two equal portions (0.06 mL each time) to the diluted solution of the product (0.8 mL DCM/methanol 50/50 solvent mixture was used each time) followed by the removal of the solvent by rotary evaporation. The addition of solvent and rotary evaporation was repeated ten times to allow for complete reaction. Doxorubicin-coupled polymer conjugate (p-Dox) was prepared similarly by reacting (azidoPEG5)2-EDTA-(GFLG-Dox)2 (4) (0.03718 g; 34.8 mM in 0.4 mL of methanol) with DBCO-PEG6-DBCO (0.01252 g; 34.8 mM in 0.4 mL of DCM).

2.4. Molecular weight analysis

The molecular weights of the drug-coupled polymer conjugates (p-Gem and p-Dox) were determined by size-exclusion chromatography (SEC) equipped with multiple angle light scattering and refractive index (concentration) detectors.47 The chromatographic system consisted of an isocratic pump (Agilent G1310B, Palo Alto, CA), well-plate autosampler (Agilent G1329A), and TSKgel Guard Alpha (18345; 6.0 mm ID × 4.0 cm, 13 μm) and TSKgel Alpha-3000 (18340; 7.8 mm ID × 30 cm, 7 μm) columns (Tosoh Bioscience LLC, King of Prussia, PA). The size exclusion column was connected in-line to a light scattering detector (DAWN HELEOS-II, 690 nm laser, Wyatt Technology, Santa Barbara, CA) and a refractive index detector (Optilab T-rEX, 658 nm laser, Wyatt Technology, Santa Barbara, CA). The isocratic mobile phase was DMF with 10 mM LiCl at a flow rate of 1 mL/min. Solutions of the drug-coupled polymer conjugates (100 μL; 20 mg/mL prepared in DMF with 10 mM LiCl) were injected into the chromatographic system.

The specific refractive index increment (dn/dc) values, representing the change in the refractive index in response to a change in concentration, were measured using calibration standards (0.1, 0.2, 0.4, 0.8, and 1 mg/mL) prepared from 20 mg/mL stock solutions of each polymer-drug conjugate. Pure solvent (1 mL, DMF with 10 mM LiCl) was injected first to produce the pure solvent baseline. Next, 800 μL of each sample (starting with the lowest concentration) was injected. After all samples had been injected, pure solvent was injected for a second baseline determination. ASTRA (v6.1.2.84, Wyatt Technology) was used to calculate dn/dc (Supporting Information, Figure S16). Multiple angle light scattering normalization constants were determined using a 30 kDa polystyrene standard (PS 80317, Pressure Chemical Co., Pittsburgh, PA) prepared at 4 mg/mL in 10 mM LiCl in DMF.

2.5. Drug loading studies

2.5.1. Evaluation of gemcitabine content in p-Gem

Gemcitabine loading in p-Gem was measured by ultraviolet-visible (UV-vis) spectroscopy. UV-Vis spectra of 100 μg/mL solutions of p-Gem (prepared using 90% v/v DMF in water) were recorded using Horiba Duetta spectrometer (HORIBA Scientific, Piscataway, NJ). The spectra were collected from 200–400 nm at a 2 nm step increment, 0.05 second integration time, and 5 nm band pass against 90% v/v DMF in water as the reference. All samples were measured in quartz cuvettes (path length b = 10 mm, Hellma, Plainview, NY). Calibration standards (1 to 75 μg/mL) prepared from a stock solution of gemcitabine HCl (1 mg/mL in 90% v/v DMF in water) were used to obtain the calibration curve used for the determination of gemcitabine content in p-Gem based on the intensity counts at 268 nm (Supporting Information, Figure S17).

2.5.2. Evaluation of doxorubicin content in p-Dox

Doxorubicin loading in p-Dox was measured by fluorescence spectroscopy using Horiba Duetta spectrometer. The emission spectra of 100 μg/mL solutions of p-Dox (prepared using 90% v/v DMF in water) at 470 nm excitation wavelength were collected from 550 to 700 nm at an emission increment of 0.5 nm, 0.05 second integration time, and 5 nm excitation/emission band pass against 90% v/v DMF in water as the reference. Calibration standards (0.5 to 20 μg/mL) prepared from a stock solution of doxorubicin HCl (1 mg/mL in 90% v/v DMF in water) were used to obtain the calibration curve used for the determination of doxorubicin content in p-Dox based on the emission intensity counts at 595 nm (470 nm excitation) (Supporting Information, Figure S18).

2.6. Evaluation of cathepsin B-catalyzed cleavage

Cathepsin B-catalyzed cleavage of p-Gem was assessed in vitro by incubation of p-Gem solution with exogenous cathepsin-B following the method of Jin et al.48 with slight modifications. Freshly prepared solutions of 1,4-dithiothreitol (DTT) (30 mM; 24 μL) and EDTA disodium salt dihydrate (15 mM; 12 μL) were added to cathepsin B enzyme solution (0.114 nM; 10 μL), and incubated at 37 °C for 10 minutes to activate the enzyme. The cleavage medium was prepared by adding Tween 20® (0.5% v/v) and DMSO (1% v/v) to phosphate buffer (pH 5.0). p-Gem (0.33 mg/mL) was incubated with the activated enzyme mix at 37 °C with continuous 360º rotation at 10 rpm using a fixed angle tube rotator (Thermo Fisher Scientific, Pittsburgh, PA). A similar reaction without the enzyme mix was set up as a control. Samples (100 μL) were withdrawn from the test and control reactions at intervals (0 – 24 h), diluted with acetonitrile (400 μL) to precipitate the enzyme, and analyzed by HPLC (Agilent 1100 Series LC C-18 column (250 × 4.6 mm, 5 mm particle size); diode array detector; mobile phase gradient (acetonitrile: water): 2% v/v acetonitrile increased to 95% v/v acetonitrile; flow rate: 1 mL/min; run time: 15 minutes). The appearance of free gemcitabine and the disappearance of p-Gem was determined by monitoring the HPLC chromatogram peak areas at the characteristic retention times of the pure drug and p-Gem.49

A similar approach was used for the doxorubicin-coupled polymer conjugate (p-Dox) by incubating p-Dox at a concentration of 0.68 mg/mL with the enzyme mix at 37 °C. Samples (200 μL) were withdrawn from the test and control reactions at intervals (0 – 24 h), diluted with acetonitrile (400 μL) and analyzed by HPLC. The appearance of the free drug and the disappearance of p-Dox was determined by monitoring the HPLC chromatogram peak areas at the characteristic retention times of the pure drug and conjugate.

3. Results and discussion

3.1. Synthesis of α-ω-bis-azide-terminated bifunctional drug-coupled monomers

We synthesized two α-ω-bis-azide-terminated drug-coupled monomers each containing gemcitabine and doxorubicin, respectively, using an EDTA derivative, (azido-PEG5)2-EDTA acid (1), as the monomer starting material. Our choice of using an EDTA derivative for the synthesis of PDC was informed by relevant background information. EDTA is FDA-approved chelating agent for the treatment of metal poisoning.50 It is usually converted into its dianhydride, EDTA dianhydride, for the synthesis of other organic compounds like polymers and hydrogels.51 EDTA dianhydride is biodegradable52 and it is used to introduce carboxylic acid groups in a chemical reaction and cross-linking polymers.52,53 Suárez del Pino & Kolhatkar43 synthesized an enzymatically degradable EDTA-based dendritic polymer for the delivery of a potent cytotoxic agent to prostate cancer cells by incorporating GFLG in the polymer backbone. The dendrimer-drug conjugate was plasma-stable and drug release from it was triggered by specific cleavage of GFLG by cathepsin B and subsequent degradation of the polymer backbone.43

The schematic of the synthesis of (azido-PEG5)2-EDTA acid (1) is reported in Scheme 1. (Azido-PEG5)2-EDTA acid (1) provided the azide homo-bifunctional groups needed for SPAAC-mediated step-growth polymerization for PDC synthesis in the presence of DBCO-PEG6-DBCO, a dibenzoazacyclooctyne (DBCO) homo-bifunctional monomer.29,30,34 It also provided the pendant carboxylic groups essential for further amide bond formation with other building units of the drug-coupled monomers.

Scheme 1.

Scheme 1.

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Synthesis of (azido-PEG5)2-EDTA acid (1) by one-step amide bond formation between azido-PEG5-amine and EDTA dianhydride.

GFLG, a cathepsin B-sensitive tetrapeptide linker, was incorporated in the monomer starting material by SPPS (Scheme 2). We employed SPPS for the synthesis of (azidoPEG5)2-EDTA-(GFLG)2 (2), the GFLG-bearing polymer backbone intermediate, because of the ease of synthesis facilitated by SPPS. The FT-IR spectrum of (azidoPEG5)2-EDTA-(GFLG)2 (2) showed azide asymmetric stretching frequency at 2106.98 cm−1, indicating that the terminal azide groups present on (azido-PEG5)2-EDTA acid (1) remained intact after its coupling to GFLG (Supporting information, Figure S5).

Scheme 2.

Scheme 2.

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Synthesis of (azidoPEG5)2-EDTA-(GFLG)2 (2) by SPPS method.

The azide-terminated homo-bifunctional gemcitabine-coupled monomer (azidoPEG5)2-EDTA-(GFLG-Gem)2 (3) was synthesized from gemcitabine HCl and (azidoPEG5)2-EDTA-(GFLG)2 (2) using HATU as the coupling reagent (Scheme 3). Similarly, (azidoPEG5)2-EDTA-(GFLG-Dox)2 (4), the second azide-terminated homo-bifunctional drug-coupled monomer, was synthesized from doxorubicin HCl and (azidoPEG5)2-EDTA-(GFLG)2 (2) using IIDQ as the coupling reagent (Scheme 4). Although the HATU coupling reaction occurs more rapidly, we selected IIDQ as the coupling reagent for doxorubicin coupling because IIDQ forms only amide bonds and can be used with unprotected hydroxy groups54, minimizing side reactions with the multiple hydroxyl groups on doxorubicin.

Scheme 3.

Scheme 3.

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Synthesis of α-ω-bis-azide-terminated bifunctional gemcitabine-coupled monomer, (azidoPEG5)2-EDTA-(GFLG-Gem)2 (3).

Scheme 4.

Scheme 4.

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Synthesis of α-ω-bis-azide-terminated bifunctional doxorubicin-coupled monomer, (azidoPEG5)2-EDTA-(GFLG-Dox)2 (4).

The analytical HPLC chromatogram of (azidoPEG5)2-EDTA-(GFLG-Gem)2 (3) showed a single peak (Supporting information, Figure S8). A single peak was similarly seen on the analytical HPLC chromatogram of (azidoPEG5)2-EDTA-(GFLG-Dox)2 (4) (Supporting information, Figure S13). This indicates that the pure compounds were isolated. The terminal azide groups present on (azidoPEG5)2-EDTA-(GFLG)2 (2) also remained intact following coupling with either gemcitabine (Supporting information, Figure S9) or doxorubicin (Supporting information, Figure S13), demonstrating the robustness of the polymer backbone intermediate.

The α-ω-bis-azide-terminated bifunctional drug-coupled monomers (compounds 3 and 4) can undergo self-accelerating step-growth polymerization in the presence of a double-strained alkyne monomer55 to generate high molecular weight PDCs. Additionally, the resulting PDCs are expected to have high drug loading because the drug is already incorporated in the starting polymer precursor. By predetermining the feed ratio of drug-coupled monomers, more controlled drug conjugation to the polymer backbone has been achieved with high drug loading.7 For instance, a controlled conjugation of poly (lactide) to paclitaxel resulted in poly (lactide)-paclitaxel conjugates with predefined drug loading.56

3.2. Synthesis of gemcitabine- and doxorubicin-coupled polymer conjugates

The α-ω-bis-azide-terminated bifunctional drug-coupled monomers underwent SPAAC-mediated step-growth polymerization in the presence of homo-bifunctional DBCO-PEG6-DBCO monomer to generate high molecular weight PDCs via click chemistry (Schemes 5 and 6). Stock solutions of DBCO-PEG6-DBCO was added in aliquots to stock solutions of (azidoPEG5)2-EDTA-(GFLG-Gem)2 (3) and (azidoPEG5)2-EDTA-(GFLG-Dox)2 (4) in different reactions to synthesize gemcitabine- and doxorubicin-loaded polymers, respectively. Similar to published reports34,35, solvent evaporation after the addition of each aliquot resulted in rapid polymerization as a result of an increase in the concentrations of the reactants as the solvent evaporates.34

Scheme 5.

Scheme 5.

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Synthesis of gemcitabine-coupled polymer conjugate (p-Gem) by SPAAC reaction between the homo-bifunctional DBCO-PEG6-DBCO and (azidoPEG5)2-EDTA-(GFLG-Gem)2 (3) monomers.

Scheme 6.

Scheme 6.

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Synthesis of doxorubicin-coupled polymer conjugate (p-Dox) by SPAAC reaction between the homo-bifunctional DBCO-PEG6-DBCO and (azidoPEG5)2-EDTA-(GFLG-Dox)2 (4) monomers.

FT-IR analysis showed that the azide functional group of (azidoPEG5)2-EDTA-(GFLG-Gem)2 (3) (azide asymmetric stretching frequency at 2111.21 cm−1) was no longer present in p-Gem (Figure 1). Similarly, the azide functional group of (azidoPEG5)2-EDTA-(GFLG-Dox)2 (4) (azide asymmetric stretching frequency at 2106.97 cm−1) was not present in p-Dox (Figure 2). The absence of the azide functional groups indicated that all the azide-containing monomers (compounds 3 and 4) had been used up in the formation of higher molecular weight molecules.29

Figure 1.

Figure 1.

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Overlaid FT-IR spectra of DBCO-PEG6-DBCO, (azidoPEG5)2-EDTA-(GFLG-Gem)2 (3), and p-Gem. The absence of azide asymmetric stretching frequency at 2111.21 cm−1 showed that the azide-containing starting monomer (compound 3) had been used up.

Figure 2.

Figure 2.

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Overlaid FT-IR spectra of DBCO-PEG6-DBCO, (azidoPEG5)2-EDTA-(GFLG-Dox)2 (4), and p-Dox. The absence of azide asymmetric stretching frequency at 2106.97 cm−1 showed that the azide-containing starting monomer (compound 4) had been used up.

In our opinion, this work is the first report of the synthesis of PDCs using SPAAC-mediated step-growth polymerization. The use of a second drug was essential to validate PDC synthesis using the novel SPAAC-mediated polymerization approach for the synthesis of PDCs. The advantages of our approach compared to other reported methods for the synthesis of PDCs for anti-cancer drug delivery include modularity, absence of an initiator or catalyst, rapid progression under mild conditions, the ease of synthesis of the monomers, being amenable to different spacers and drugs, and the use of highly specific copper-free click chemistry.

3.2.1. Molecular weight analysis

The use of size exclusion chromatography (SEC) with multiple detectors which allows continuous monitoring of multiple angle light scattering (MALS) and refractive index (RI) provides a more accurate analysis of polymer molecular weight, independent of their retention time during SEC.57,58 The molecular weights of the synthesized gemcitabine and doxorubicin PDCs were measured using SEC coupled with MALS and RI detectors (SEC/MALS-RI). A summary of the measured molecular weights (Mw, Mn and Mp) and polydispersity indices (Mw/Mn) of the PDCs are given in Table 1.

Table 1.

Molecular weight analysis of synthesized PDCs by SEC-MALS/RI

PDCs Mw (kDa) Mn (kDa) PdI Mp (kDa)
p-Gem 40.18 29.33 1.37 40.37
p-Dox 1800 1782.18 1.01 1920
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Mw = weight-average molecular weight; Mn = number-average molecular weight; PdI = polydispersity index = Mw / Mn; Mp = molecular weight of the most abundant species based on the RI signal.

The doxorubicin-coupled polymer (p-Dox) had very large molecular weights (~ 1800 kDa), whereas the gemcitabine-coupled polymer (p-Gem) had much lower molecular weights (~ 40 kDa) (Figure 3). The reason for the differences in molecular weights obtained for p-Dox compared to p-Gem is not known, but differences in the molecular weights of the bis-azide monomers as a result of coupling to drugs of different molecular weights and different physicochemical characteristics could have contributed to the varying molecular weights. These large differences in molecular weights have been reported in literature for polymerizations using SPAAC. For instance, Xiang et al. reported the synthesis of polymers with Mw as low as 72.05 kDa and as high as 706.1 kDa via SPAAC-mediated step-growth polymerization reaction between sym-dibenzo-1,5-cyclooctadiene-3,7-diyne and bis-azide monomers by using different stoichiometric ratios of the monomers.41 Using the same SPAAC-mediated step-growth polymerization method, Meichsner et al. reacted bis-dibenzoazacyclooctyne with different diazide compounds to prepare polymers with molecular weights between 20.1 kDa and 89.5 kDa.35

Figure 3.

Figure 3.

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SEC chromatograms of the polymer conjugates of gemcitabine (left) and doxorubicin (right) showing the light scattering (continuous green line) and differential refractive index (dotted blue line) following SEC with MALS and RI detection. The molecular weight distribution is shown in red and was calculated using the measured dn/dc values.

The polydispersity index (PdI), defined as the ratio of weight-average molecular weight (Mw) to number-average molecular weight (Mn) (i.e. Mw/Mn), describes the molecular weight distribution of polymers.59,60 PdI values < 1.5 were obtained for the PDCs, indicating a narrow molecular weight distribution. We also determined the molecular weights of the most abundant species (Mp) of PDCs using the RI signal, a concentration detector. Our data show that the Mp values are close to the Mw values obtained for the PDCs (Table 1). This suggests that the polymerization method used in this work results in PDCs with relatively narrow molecular weight distribution.

3.2.2. Percent drug loading

Gemcitabine and doxorubicin loading in the PDCs were measured by UV-vis and fluorescence spectroscopy, respectively, using triplicate sample solutions of the PDCs in 90% v/v DMF in water. The drug loading was calculated for each sample using calibration standards obtained from each pure drug (Supporting Information, Figures S17 and S18) and normalized to the total PDC weight to yield the weight percent of each drug in the PDCs. The UV-vis spectra of pure gemcitabine HCl and p-Gem showed similar maximum wavelength of absorption at ~270 nm, indicating gemcitabine coupling to polymer (Supporting Information, Figure S19). Similarly, the fluorescence spectra of pure doxorubicin HCl and p-Dox showed identical spectra with maximum emission intensity at ~590 nm when excited at 470 nm (Supporting Information, Figure S20), indicating doxorubicin conjugation in the PDC. Figure 4 summarizes the weight percent of drugs in each PDC sample. Gemcitabine loading in the polymer conjugates was greater than doxorubicin loading. This could be due to the smaller molecular size of gemcitabine (263 Da) compared with doxorubicin (543 Da).

Figure 4.

Figure 4.

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Percent drug loading (% Wt.) in the polymer-drug conjugates as determined by UV-vis (for gemcitabine) and fluorescence spectroscopy (for doxorubicin). (n = 3)

Another advantage of our approach in addition to ease of synthesis, modularity, rapid progression under mild temperature, and absence of an initiator or catalyst, is high drug loading. For instance, the loading of gemcitabine and doxorubicin in our PDCs was higher than that obtained in other studies that used poly (2-ethyl-2-oxazoline) (<7 % wt. doxorubicin)33, HPMA copolymers (6.5 % wt. doxorubicin)19; (7.7 % wt. gemcitabine)3; (<8 % wt. gemcitabine)26; (<13 % wt. gemcitabine)27, and PEG (<8.5 % wt. doxorubicin)61 as the polymer backbone.

3.2.3. In vitro cathepsin B-catalyzed cleavage and drug release

Drug release from the PDCs is essential for eliciting the pharmacological effect of the coupled chemotherapeutic drugs. GFLG was selected as the linker between the drugs and the polymer backbone because it is widely used as a substrate for cathepsin B, which is highly overexpressed in many solid tumors.9,10 It is also stable in the plasma and selective for cathepsin B.18–20 The cathepsin B-catalyzed cleavage of p-Gem and p-Dox was assessed by monitoring the HPLC chromatogram peak areas at the characteristic retention times of the pure drugs and the PDCs following incubation of the PDCs in phosphate buffer (pH 5.0) at 37 °C in the presence of exogenous cathepsin B. pH 5.0 was used to simulate acidic tumor pH. In addition, cathepsin B is enzymatically active at acidic pH.62,63

Evaluation of gemcitabine cleavage from the gemcitabine-coupled polymer conjugate (p-Gem) in the presence of exogenous cathepsin B, showed an initial rapid release of gemcitabine within 2 h, followed by a gradual release over 24 hours (Figure 5, left). In addition, analysis of the cleavage test solution by mass spectrometry showed that free gemcitabine was released. A peak at m/z 526.3, corresponding to [2M + H]+ (calculated 527.2), indicating the formation of a non-covalent dimer in the gas phase,64 was found (Supplementary information, Figure S21). The analytical HPLC chromatogram of p-Gem that was incubated without cathepsin B (control) showed a peak retention time 7.3 min but was absent in the test, which showed a new peak retention time 2.9 min, which may correspond to gemcitabine (Supplementary information Figure S22 (a, b)). This showed that the released gemcitabine observed in the cleavage experiment was due to cathepsin B-catalyzed cleavage of p-Gem.

Figure 5.

Figure 5.

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Left: Cathepsin B-catalyzed release of gemcitabine (Gem) from the gemcitabine-coupled polymer (p-Gem) in the presence (blue line) and absence (red line) of exogenous cathepsin B at pH 5.0. The percent gemcitabine released at the different time points was calculated from the peaks of free gemcitabine versus polymer-bound gemcitabine monitored by a diode array detector at 275 nm. Right: Cathepsin B-catalyzed release of a doxorubicin fragment from the doxorubicin-coupled polymer (p-Dox) in the presence (blue line) and absence (red line) of exogenous cathepsin B at pH 5.0. The percent doxorubicin fragment released at the different time points were calculated from the peaks of the doxorubicin fragment versus polymer-bound drug monitored by a diode array detector at 275 nm.10,65,66

For the doxorubicin-coupled polymer conjugate (p-Dox) in the presence of exogenous cathepsin B, a red film was observed on the wall of the glass vial containing the cleavage reaction mixture at 3 h. In addition, a new peak (retention time 3.8 min) was observed when the cleavage test solution was monitored at 275 nm wavelength at 3 h but was absent in the control with no cathepsin B (Supplementary information, Figure S22 (c, d)). It was also observed that the percent release of this compound (retention time 3.8 min) increased over 24 hours (Figure 5, right).

Doxorubicin is reported to form a precipitate in buffers, such as PBS, as a result of the formation of doxorubicin dimers.67 Also, the presence of a hydrophobic anthraquinone ring and a hydrophilic amino sugar moiety in the structure of doxorubicin, suggests that doxorubicin is amphiphilic.67 The red film could be due to the precipitation of the hydrophobic doxorubicin aglycone moiety while the peak at 3.8 min may be due to the hydrophilic amino sugar moiety following hydrolysis of the released doxorubicin in PBS (Supplementary information, Figure S23).68 To support this hypothesis, acetonitrile was added to the cleavage test solution at 30 h to re-dissolve the red film that had formed on the container wall and the sample was subjected to mass spectrometry analysis. The ESI+-MS spectrum of re-dissolved p-Dox cleavage test solution at 30 h (Supplementary information, Figure S24) showed mass corresponding to the non-covalent dimer of the hydrophilic doxorubicin amino sugar moiety (m/z calculated for C12H25N2O6 [2M + H]+ = 293.2, found 293.1). The m/z at 481.3 and 499.3 also corresponded to the molecular weight of the doxorubicin aglycone fragmentation products.68

4. Conclusion

GFLG-containing α-ω-bis-azide-terminated bifunctional gemcitabine- and doxorubicin-coupled monomers were synthesized for the development of cathepsin-B activatable PDCs of both anticancer drugs. The α-ω-bis-azide-terminated bifunctional drug-coupled monomers were prepared from a linear hydrophilic EDTA derivative via simple synthetic steps and copolymerized with a DBCO-PEG6-DBCO homo-bifunctional monomer using SPAAC-mediated step-growth polymerization. DBCO reacts with azide groups in a highly specific copper-free click chemistry, thus, the resulting PDCs were easily isolated following solvent removal by rotary evaporation with no by-product. In addition, the SPAAC-mediated step-growth polymerization method of synthesis of the PDCs did not require the use of high temperature, initiator, or catalyst.

The novel design of the PDCs may facilitate selective delivery and passive targeting of cancer by the EPR effect and active targeting of cathepsin B enzyme overexpressed selectively in many solid cancers. The prepared PDCs have molecular weights greater than 40 kDa (renal filtration threshold) and have higher drug loading when compared to other reported approaches. Thus, they are suitable for passive targeting of cancer by the EPR effect and selective release of higher loads of cytotoxic drugs in the tumor microenvironment. The PDCs also showed cathepsin B-catalyzed cleavage and drug release at pH 5.0, and no drug release in the absence of cathepsin B within the period evaluated. We envision that the use of SPAAC-mediated polymerization for the syntheses of tumor-targeted PDCs of gemcitabine and doxorubicin as model drugs will facilitate the accumulation of these potent anticancer agents in tumors leading to greater cytotoxicity in the tumor compared with free drugs and reduced toxicity to healthy cells.

Supplementary Material

Supinfo

Acknowledgement:

This work was supported, in part, with federal funds from the National Cancer Institute, National Institutes of Health, under contract no. 75N91019D00024. The formulation described herein was accepted into the Assay Cascade characterization program of the Nanotechnology Characterization Laboratory (NCL) of the Frederick National Laboratory for Cancer Research. The NCL provides a free characterization service for cancer-related nanomedicine formulations, available to the public by application (https://www.cancer.gov/nano/research/ncl). The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

Footnotes

Supporting Information: Analytical HPLC chromatograms, FT-IR spectra, 1H NMR spectra, and mass spectrometry spectra of select compounds; specific refractive index (dn/dc) plots; UV-Vis spectra, fluorescence spectra, and calibration curves for drug content evaluation.

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