Cyclodextrin polymers as carriers for the platinum-based anticancer agent LA-12

Abstract

Polymeric nanoparticles containing cyclodextrins are currently undergoing clinical trials as nanotherapeutics. In this context, we have synthesized high and low molecular weight β-cyclodextrin polymers and functionalized them with folate to improve their selectivity for cells overexpressing the folic acid receptor. Inclusion complexes of the unfunctionalized and FA-functionalized polymers with the antitumour complex cis–trans–cis-[PtCl₂(CH₃CO₂)₂(adamantyl-NH₂)(NH₃)] (LA-12) have been tested on tumour cells. In the presence of the high molecular weight polymers, LA-12 exhibited IC₅₀ values significantly lower than that of LA-12 alone in MDA-MB-231 cells. The drug nanocarriers investigated do not appear to take significant advantage of the presence of folate residues, possibly because of the reduced accessibility of folate included in cyclodextrin moieties by the receptor or low degree of functionalization of the polymers. However the non-covalent approach, based on inclusion complexes with cyclodextrin polymers, looks very promising for improving the performance of LA-12.

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Introduction

Cyclodextrins (CyDs) are cyclic oligomers of α-1,4 linked D(+)-glucopyranosyl units. The most common CyDs are α-, β-, and γ-CyDs with six, seven, or eight glucose units, respectively. The complexing properties of CyDs have been widely investigated and, as a result, CyDs have found several applications in the pharmaceutical, textile, and food industries.^1–3

By replacement of CyD hydroxyl groups with other functional groups it has been possible to build multifunctional systems and to widen markedly the field of application of CyDs. Thus, over the years, CyD covalent conjugates with amino acids, amines and aromatic systems have been reported.^4–7 Fascinating compounds, including enzyme mimics, abiotic receptors, and antiaggregant agents, have been obtained by shaping the hydrophobic nature and size of the CyD cavity so as to match the specific requirements of the ad hoc attached molecule.⁸

Cyclodextrin-based carriers, besides their ability to encapsulate guest molecules, can also improve the stability of the drug and efficiently regulate the rate of drug release.^9,10 Thus a variety of nanosystems, spanning from organic polymers to inorganic nanoparticles, have been decorated with CyDs.^11–13 CyD polymers can exhibit a dramatic enhancement of the inclusion ability towards various guests.^8,9,14,15 A successful example of linear CyD polymers designed specifically to overcome some limitations in the systemic transport of a drug is Cyclosert. CRLX-101 is a nanoparticle that consists of camptothecin (CPT) conjugated to CyD polymers based on the Cyclosert delivery platform. CRLX-101 is presently undergoing clinical trials for cancer nanotherapy.¹⁶ CALAA-01 is a cyclodextrin-containing polymer, which is a nanoformulation carrying siRNA for targeted RNA interference (RNAi) therapy.¹⁷ These nanoparticles are prepared via the self-assembly of cyclodextrin-containing polymers in the presence of nucleic acids. CALAA-01 is currently undergoing a phase II clinical trial against melanoma; it has demonstrated evidence of successful RNAi in humans.¹⁸

To increase their drug loading features, CyD polymers have also been modified through the incorporation of anionic or cationic groups.¹⁹ CyD polymers have been modified with choline or carboxyl moieties and the effect on the upload of charged drugs has been investigated.¹⁹ More recently, the synthesis of oligomers has also been exploited since drug carriers with low molecular weight can have some advantages such as easy excretion by renal tubules without degradation.²⁰

For targeting purposes, the isoform α of the folate receptor (FR) has been identified as an attractive tumour marker which is overexpressed on the surfaces of several cancer cells.²¹ Therefore, folic acid (FA, vitamin B9, (2S)-2-[(4-{[(2-amino-4-hydroxypteridin-6-yl)methyl]amino}phenyl)formamido] pentanedioic acid) has emerged as a valuable cell-targeting moiety.²² It has also been demonstrated that the FR recognises folic acid derivatized at the γ-carboxylate.²³ Derivatization with FA has been applied also to β-CyDs,^24–31 and recently we have synthesized new FA derivatives of CyDs which were able to significantly improve the cytotoxicity of LA-12 (cis–trans–cis-[PtCl₂(CH₃CO₂)₂(adamantyl-NH₂)(NH₃)], Fig. 1), a complex of Pt(IV) with 1-adamantylamine (AMA), towards cancer cells that overexpress the FR.³² LA-12 acts as a pro-drug of an analogue of cisplatin,³³ the latter representing a cornerstone in present-day chemotherapy. Significant side effects, such as nephrotoxicity, neurotoxicity, and tumour resistance considerably limit the potential of cisplatin in cancer therapy. For these reasons, big efforts have been devoted to the development of novel platinum-based chemotherapeutics with less severe side effects and which are active even on cisplatin-resistant cancer types. LA-12 is a promising new platinum complex that has reached phase I clinical trials. It can be administered orally (mouse models) and is active against intrinsically cisplatin-resistant ovarian adenocarcinoma.^34–36 The inclusion of LA-12, poorly soluble in water (about 0.03 mg mL⁻¹), into β-CyDs has been patented³⁷ as a new pharmaceutical formulation suitable for LA-12 administration by injection. Indeed, the adamantyl group has high affinity for the β-CyD cavity with a K_a of about 5000 M⁻¹.^38–40

Fig. 1 Sketched structure of oCyDFA, folic acid and LA-12.

The promising results obtained with β-CyD-FA conjugates as carriers for LA-12 ³² prompted us to extend the investigation to β-CyD polymers and oligomers functionalized with FA and to compare them with analogous systems which are FA free (Fig. 1). In particular, we synthesized FA conjugates starting from a commercial polymer (pCyDFA) and a synthetic amino oligomer (oCyDFA); furthermore we tested the cytotoxicity of their inclusion complexes with LA-12 in FR(+) and FR(−) cell lines.

Results and discussion

oCyD was synthesized starting from β-CyD. The polymerization reaction was carried out under basic conditions using epichlorohydrin (ECH) as the cross-linking agent. The low ECH/monomer ratio (ECH/CyD = 5) ensured the obtainment of an oligomer.^41,42 oCyD was characterized with NMR and light scattering (Fig. 1S–5S†).

The NMR spectrum of oCyD (Fig. 1S†) showed broad peaks, as typically observed for these classes of compounds. From integration of the Hs-1 signal of CyD and the broad signal at 4.2–3.2 ppm, it was possible to estimate the ratio between the number of CyDs and that of the cross-linker chains in the oligomer. The obtained value suggests that the oligomer has a CyD content of about 65%. DLS measurements show that oCyD forms nanoparticles of about 3.8 nm diameter at 3 mg mL⁻¹ (pH 7.4) (Fig. 2S†). The average molecular weight (M_w) of oCyD was determined through light scattering measurements as reported for oCyDNH₂.⁴² The plot of K_c/R_θ vs. the concentration of oligomer is shown in Fig. 3S.† The intercept of the straight line was used to determine the M_w value, which was found to be 12 ± 1 kDa. This value corresponds to an average number of 7 cavities per molecule.

As for commercial pCyD, DLS measurements show that pCyD forms nanoparticles of about 8.5 nm diameter at 3 mg mL⁻¹ (Fig. 2S†). pCyD was tosylated using the same procedure used for the synthesis of 3-tosyl-β-cyclodextrin.⁴³ The tosylate was converted into an amino group by the synthetic procedure reported for 3A-amine-3A-dideoxy-2A(S),3A(R)-β-cyclodextrin.⁴³ Both the amino polymer and the amino oligomer were functionalized with folic acid-N-hydroxysuccinimidyl ester.

The ¹H NMR spectrum of pCyDFA (Fig. 4S†) shows broad bands as typically found in the case of polymers. From integration of the signals of the folic acid moiety and that of the Hs-1 of CyD it is possible to estimate that 12–15% of CyD cavities have been functionalized with FA.

A similar amount of FA was found when oCyDNH₂ was conjugated with FA.

Interaction with LA-12

The water solubility of LA-12 increased markedly both in the presence of the polymer and oligomer, in keeping with its inclusion in the CyD cavity. The inclusion of LA-12 into CyD polymers and oligomers was investigated using NMR (Fig. 5S†). A NOESY spectrum of a mixture of pCyDFA and LA-12 (1 : 23 molar ratio) is shown in Fig. 2. The NOE cross peaks between the adamantane protons and the CyD protons show the formation of the inclusion complex. In addition, the peaks due to the adamantane residue of LA-12 are also very broad as an effect of its inclusion in the polymer. A similar behavior was found for oCyDFA.

Fig. 2 NOESY spectra of pCyDFA/LA-12 in D₂O (500 MHz).

Antiproliferative activity (MTT assay)

Cell proliferation assays were performed on cancer cell lines differing in their degree of FR expression (A2780, MDA-MB-231, MCF-7).²¹ MCF-7 cells were used as a negative control.

pCyDFA/LA-12, oCyDFA/LA-12, pCyD/LA-12 and oCyD/LA-12 complexes were tested in all cell lines (Table 1). Two host/LA-12 molar ratios were tested. The host concentration was expressed as “CyD cavity” concentration, calculated on the basis of the M_w and β-CyD% content of the hosts. pCyD or oCyD were not toxic for cells up to a concentration of 50 μM. The toxicity of CyD/LA-12 is also reported in Table 1 for comparison. CyD/LA-12 shows the same IC₅₀ as LA-12.

Table 1 IC₅₀s (μM) of CyD polymers/LA-12 in human tumour cells

Compounds	Cell lines
	A2780	MDA-MB-231	MCF-7
a The values represent the mean ± SD of 4–10 data.b p < 0.001 vs. LA-12.c p < 0.01 vs. pCyD/LA-12 (1 : 1).d p < 0.001 vs. LA-12 and pCyD/LA-12 (1 : 1).e p < 0.05 vs. LA-12.f p < 0.01 vs. LA-12.g p < 0.02 vs. LA-12.h p < 0.01 vs. pCyD/LA-12 (5 : 1).i p < 0.001 vs. pCyD/LA-12 (5 : 1).
LA-12	0.59 ± 0.15^a	2.40 ± 0.45	4.29 ± 0.86
pCyDFA/LA-12 (5 : 1)	0.24 ± 0.05^b^,^c	0.78 ± 0.24^d	5.55 ± 0.76
oCyDFA/LA-12 (5 : 1)	0.66 ± 0.18	1.41 ± 0.12^g	5.62 ± 0.45
oCyDFA/LA-12 (1 : 1)	0.36 ± 0.13^e	2.86 ± 0.44	6.91 ± 1.68
pCyD/LA-12 (1 : 1)	0.44 ± 0.08^e	0.67 ± 0.18^d	4.04 ± 0.51
pCyD/LA-12 (5 : 1)	0.34 ± 0.10^f^,^h	0.53 ± 0.07^d^,^i	5.05 ± 1.01
oCyD/LA-12 (1 : 1)	0.41 ± 0.11	2.87 ± 0.52	7.27 ± 2.42
oCyD/LA-12 (5 : 1)	0.72 ± 0.25	1.63 ± 0.14^f	4.53 ± 0.53
CyD/LA-12 (5 : 1)	0.58 ± 0.16	2.79 ± 0.20	5.51 ± 0.57
pCyD	>50	>50	>50

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As shown in Table 1, MCF-7 cells are the least sensitive to LA-12. In A2780 and MDA-MB-231 cells, pCyDFA/LA-12 and pCyD/LA-12 complexes were the most active and showed an increased toxicity, in comparison to LA-12, of 59% and 68%, respectively. Unlike pCyD derivatives, oCyD and oCyDFA derivatives did not significantly increase the toxicity of LA-12 in A2780 and MCF-7 cells, while only a slight decrease in IC₅₀ was observed in MDA-MB-231 cells when using a higher oCyD/LA-12 molar ratio. It is noteworthy that the complexes of LA-12 with high molecular weights are significantly more active than the complexes with CyD oligomers. Furthermore, MDA-MB-231 became more sensitive to LA-12 in the presence of polymers.

Experimental

Materials and methods

All chemicals obtained from commercial sources were used without further purification. The water soluble polymer pCyD (92 kDa, 70% of CyD) was purchased from Cyclolab. 3A-Amino-3A-dideoxy-2A(S),3A(R)-β-cyclodextrin and amino-oligomer (oCyDNH₂) were synthesized as described elsewhere.^41,43 Thin layer chromatography (TLC) was carried out on silica gel plates (Merck 60-F254). Carbohydrate derivatives were detected on TLC by UV light and/or by the anisaldehyde test.

CM Sephadex-25 (Sigma Aldrich) was used for column chromatography. Thin Layer Chromatography (TLC) was carried out on silica gel plates (60-F254, Merck, Darmstadt, Germany).

Folate N-hydroxysuccinimide (FA-NHS) was synthesized as reported elsewhere.⁴⁴ Stock solutions of LA-12 were prepared in DMSO. Inclusion complexes of LA-12 and CyDs were prepared by mixing the stock solution of LA-12 with aqueous solutions of CyD polymers.

NMR spectroscopy

NMR spectra were recorded at 300 K with a Varian UNITY PLUS-500 spectrometer, by using standard pulse programs from the Varian library. The 2D experiments (COSY, TOCSY, HSQC, and ROESY) were performed using 1K data points, 256 increments and a relaxation delay of 1.2 s. The spectra were referred to the solvent signal.

Synthesis of β-cyclodextrin oligomer (oCyD)

β-CyD (5 g, 4.4 mmol) in 10 mL of NaOH solution (7 M) was stirred overnight at 25 °C. ECH (1.72 mL, 22 mmol) was slowly added under stirring. After 6 h the reaction was stopped by addition of acetone. After decantation, the acetone was removed. The suspension was maintained at 50 °C overnight, then was brought to 25 °C, treated with 6 M HCl until neutral pH was reached, and filtered using ultra-filtration (molecular weight cut-off 5000). Addition of ethanol caused the precipitation of the final product, which was filtered, and freeze-dried to give a white powder. Yield: 40%.

oCyD. ¹H NMR (500 MHz, D₂O) δ (ppm): 5.20–4.82 (7H, m, H-1), 4.27–3.22 (69H, m, H-5, -6, -3, -2, -4, H of linker). Dimension (DLS): 3.8 ± 0.8 nm. Zeta potential: 2.1 ± 1 mV.

Synthesis of pCyDFA

pCyDtos. Freshly activated molecular sieves of 4 Å (0.50 g) were added to a DMF solution (6.0 mL) of pCyD (0.1 g) and p-toluenesulfonyl imidazole (Tos-Im, 22 mg) and the mixture was left under stirring for 48 h. After removal of the molecular sieves, the solvent was evaporated under vacuum and the solid washed with acetone and dried.

The solid was dissolved in water and purified by dialysis. NMR was used to determine the amount of tosyl groups.

pCyDtos was dissolved in 4 mL of water and treated with 400 mg of NaHCO₃ under stirring. After 4 h, 1 mL of NH₃ solution (28%) was added and the reaction mixture kept at 60 °C under an N₂ atmosphere for 12 h. After evaporation of the solvent, the solid was dissolved in water and purified by ultrafiltration.

The obtained amino polymer and FA-NHS (50 mg) were dissolved in dry DMF (2 mL), TEA (10 μL) was added and the reaction mixture was stirred at 25 °C for 48 h. DMF was evaporated under vacuum, and the solid residue was dissolved in water (3 mL) and purified by dialysis.

¹H NMR (500 MHz, D₂O) δ (ppm): 8.60 (bs, H-7 of FA), 7.56 (bs, H-13 and H-15 of FA), 6.62 (bs, H-12 and H-16 of FA), 5.20–4.82 (7H, m, H-1), 4.27–3.22 (69H, m, H-5, -6, -3, -2, -4, ECH and H-FA).

oCyDFA. oCyDNH₂ (100 mg) and FA-NHS (50 mg) were dissolved in dry DMF (2 mL), TEA (10 μL) was added and the reaction mixture was stirred at room temperature for 48 h. After evaporation of the solvent, the product was dissolved in water (3 mL) and purified by dialysis.

¹H NMR (500 MHz, D₂O) δ (ppm): 8.60 (bs, H-7 of FA), 7.56 (bs, H-13 and H-15 of FA), 6.62 (bs, H-12 and H-16 of FA), 5.20–4.82 (7H, m, H-1), 4.27–3.22 (69H, m, H-5, -6, -3, -2, -4, ECH and H-FA).

Refractive index increment (dn/dc) determination

Refractive index increment (dn/dc) was determined by surface plasmon resonance imaging (SPRI) using SPR imager IID apparatus (GWC Technologies, USA) coupled with a poly(dimethylsiloxane) microfluidic device as elsewhere described.⁴⁵

dn/dc determinations were carried out under flow conditions by detecting SPRI signals generated by differently concentrated oCyD and pCyD solutions. Air bubbles were properly introduced into the fluidic device with the aim of preventing the diffusion of the analyte at the interface between the solutions.⁴⁶

The SPRI apparatus was calibrated using sucrose solutions (0.0150, 0.0290, 0.0590, 0.0890, 0.1180 M in water) with a known refractive index (Fig. 6S†).⁴⁷ The obtained calibration curve was used to convert absolute changes in SPRI reflected intensity generated by differently concentrated oCyD and pCyD solutions into refractive index changes (Fig. 7S and 8S†).

dn/dc corresponds to the slope of the dependence of refractive index of a solution as a function of the solute concentration. Hence, refractive index values of oCyD and pCyD solutions deduced from the calibration curve were respectively plotted versus concentration values (concentration range 1.0–15.0 mg mL⁻¹ in Tris(hydroxymethyl)aminomethane (TRIS) buffer pH 7.4). The slopes of the straight line (dn/dc) were 0.135 ± 0.005 mL g⁻¹ for oCyD (Fig. 7Sb†) and 0.135 ± 0.007 mL g⁻¹ for pCyD (Fig. 8Sb†), respectively. The dn/dc value for pCyD is the same as that reported elsewhere for neutral CyD polymers.⁴⁸

Light scattering measurements

Dynamic light scattering (DLS) measurements were performed at 25 °C with a Zetasizer Nano ZS (Malvern Instruments, UK) operating at 633 nm (He–Ne laser). The scattered light was detected at an angle of 173°.

Weight average molecular weight (M_w) was determined with static light scattering measurements as reported elsewhere.^41,48 Samples were studied at different concentrations applying the Rayleigh equation, which reports the intensity of the light scattered from the oligomer solution:

where R_θ is the ratio of scattered light to incident light of the sample; M_w is the weight average molecular weight; A₂ is the second virial coefficient; c is the polymer concentration; P_θ is the angular dependence of the sample scattering intensity; K_c is the optical constant defined as:
where N_A is Avogadro’s constant; λ₀ is the laser wavelength; n₀ is the solvent refractive index; dn/dc is the differential refractive index increment. dn/dc = 0.135 mL g⁻¹ as determined with SPR was used. Toluene was used as the reference. K_c/R_θ can be plotted as a function of c. The slope (2A₂) will give the second virial coefficient and the intercept (1/M_W) will give the molecular weight. Aqueous buffered solutions of oCyD (concentration range: 1.0–15.0 mg mL⁻¹ in 10 mM TRIS buffer, pH 7.4) were filtered with a nylon membrane filter (0.22 μm). The size distribution of the oligomer was measured during the determination of molecular weight. Triplicate measurements were carried out. Every measurement was the average of at least 12 runs.

Cell culture

Ovarian carcinoma A2780 cells were grown as monolayers in RPMI medium (Euroclone, Pero, Italy) supplemented with 10% FBS and 1% penicillin-streptomycin (Euroclone). Breast carcinoma MDA-MB-231 and MCF-7 cells were grown in DMEM medium (Euroclone) supplemented with 10% FBS and 1% penicillin–streptomycin (Euroclone).

Determination of antiproliferative activity by the MTT assay

Cell lines were plated (180 μL of a suspension of appropriate concentration) into flat-bottomed 96-well microtiter plates. After 6–8 h incubation, cells were treated with the selected complexes (five solutions, 20 μL) in a dilution ratio of 1 : 5 (starting from 10 μM concentration) and processed by the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) as described elsewhere.⁴⁹

The stock solutions of LA-12 were freshly prepared in DMSO. The final concentration of DMSO in the cell culture medium did not exceed 0.2%. LA-12 and its complexes with CyD polymers were used starting from 10 μM (referred to LA-12).

The IC₅₀ values were calculated on the basis of the analysis of single concentration–response curves, each final value being the mean of 4–10 experiments.

Student’s test was used for the statistical analysis of data.

Conclusions

Herein we reported the use of cyclodextrin-based drug nanocarriers. Two new β-cyclodextrin polymers with different molecular weights (about 90 and 12 kDa) were synthesized and functionalized with FA. By the adopted synthetic procedure the extent of modification with FA was only 15% of the cavities.

All these systems are able to include the anticancer agent LA-12 and to improve greatly its solubility in water.

While the high molecular weight polymers increased significantly the cytotoxicity of LA-12 in the cell lines tested, this was not the case for the low molecular weight systems. This is probably because pCyD nanoparticles could enhance the permeability of LA-12 across cell membranes better than oCyD. This ability could depend on the dimensions of the nanocarrier.¹

Contrary to expectations, no improvement of toxicity was found after functionalization with FA, probably because of the low amount of functionalization (15%) and/or the low accessibility of FA by the receptor. Indeed, FA can be self-included in the β-CyD cavity.³²

To the best of our knowledge, this is the first report of cyclodextrin polymers used as nanocarriers of LA-12. Furthermore, this is the first time that FA was conjugated to polymers or oligomers of cyclodextrins.

These results suggest that cyclodextrin polymers can be very promising as nanocarriers for poorly water soluble anticancer drugs.

Acknowledgements

The authors acknowledge support from the University of Bari “Aldo Moro”, the University of Catania, the Fondazione Cassa di Risparmio di Puglia (Project “Spettroscopie avanzate per lo studio delle interazioni in cellula”), the Consorzio Interuniversitario di Ricerca in Chimica dei Metalli nei Sistemi Biologici (CIRCMSB), and the Italian Ministero dell’Università e della Ricerca (PON01_01078 and FIRB RINAME RBAP114AMK). We are grateful to Dr Maurizio Losacco for performing spectroscopic measurements.

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Footnote

† Electronic supplementary information (ESI) available: NMR spectra, DLS and SPR data. See DOI: 10.1039/c5ra22398a

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