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. Author manuscript; available in PMC: 2016 May 1.
Published in final edited form as: Biomaterials. 2015 Feb 16;51:22–29. doi: 10.1016/j.biomaterials.2015.01.075

Nanoparticle-mediated delivery of a rapidly activatable prodrug of SN-38 for neuroblastoma therapy

Ivan S Alferiev 1,#, Radhika Iyer 1,#, Jamie L Croucher 1,#, Richard F Adamo 1, Kehan Zhang 1, Jennifer L Mangino 1, Venkatadri Kolla 1, Ilia Fishbein 1, Garrett M Brodeur 1, Robert J Levy 1, Michael Chorny 1,*
PMCID: PMC4361798  NIHMSID: NIHMS661153  PMID: 25770994

Abstract

Nanomedicine-based strategies have the potential to improve therapeutic performance of a wide range of anticancer agents. However, the successful implementation of nanoparticulate delivery systems requires the development of adequately sized nanocarriers delivering their therapeutic cargo to the target in a protected, pharmacologically active form. The present studies focused on a novel nanocarrier-based formulation strategy for SN-38, a topoisomerase I inhibitor with proven anticancer potential, whose clinical application is compromised by toxicity, poor stability and incompatibility with conventional delivery vehicles. SN-38 encapsulated in biodegradable sub-100 nm sized nanoparticles (NP) in the form of its rapidly activatable prodrug derivative with tocopherol succinate potently inhibited the growth of neuroblastoma cells in a dose- and exposure time-dependent manner, exhibiting a delayed response pattern distinct from that of free SN-38. In a xenograft model of neuroblastoma, prodrug-loaded NP caused rapid regression of established large tumors, significantly delayed tumor regrowth after treatment cessation and markedly extended animal survival. The NP formulation strategy enabled by a reversible chemical modification of the drug molecule offers a viable means for SN-38 delivery achieving sustained intratumoral drug levels and contributing to the potency and extended duration of antitumor activity, both prerequisites for effective treatment of neuroblastoma and other cancers.

Keywords: drug delivery, nanoparticle, prodrug, topoisomerase I inhibitor, neuroblastoma

Introduction

Nanoparticle (NP)-mediated delivery has shown promise for improving the efficacy and safety of different classes of anticancer agents. Enhanced therapeutic performance and reduced toxicity of pharmaceuticals formulated in NP are expected to result from their improved biocompatibility, potentially more favorable biodistribution modulated through adjustments in the nanocarrier design, extended presence at therapeutically effective levels and protection from hydrolytic or enzymatic inactivation of the NP-associated drug cargo [1]. However, the successful translation of experimental NP-based therapies into the clinic has been hampered by several barriers [2–4]. Among these, a challenge often underestimated in the design of cancer nanomedicines is achieving stable entrapment and retention of the drug in the submicronial carrier and subsequent release in its pharmacologically active form at the site of action, which are both key to realizing the advantages of NP-based therapeutic strategies [5]. This stems from the difficulty in overcoming the rapid drug escape driven by a partition equilibrium perturbation when the administered nanocarrier is extensively diluted in a medium with a large capacity for its cargo, as predicted theoretically [6] and recently shown experimentally in animal studies [7–9]. The prematurely released drug reassumes the characteristics of the free chemotherapeutic agent, thus precluding the potential benefit expected from the use of the NP formulation [10].

Directed changes in the drug molecular structure aimed at forming bioreversible precursors (prodrugs) with increased affinity for the carrier have been explored as a means of preventing premature drug dissociation [11–14]. However, the effective use of the prodrug approach poses an additional set of challenges, because the kinetics of prodrug release and subsequent parent compound activation need to be tightly coordinated with that of the NP biodistribution. Furthermore, when applied to metabolically unstable agents, the pharmacophore regeneration rate should also be adjusted to outcompete concurrent breakdown processes to ensure adequate recovery of the drug in its functional form.

Camptothecin and its analogs are a family of potent small-molecule topoisomerase I inhibitors with a broad spectrum of activity against adult and pediatric cancers [15, 16], whose safety, metabolic stability and therapeutic efficacy could potentially be greatly improved by using properly formulated nanocarriers [17]. In the present study, a formulation strategy integrating prodrug modification and a NP preparation method designed for producing sub-100 nm sized, bioeliminable NP was applied for nanoencapsulation and delivery of a 7-ethyl-10-hydroxy analog of camptothecin, SN-38. A derivatization approach based on phenolic ester chemistry uniquely suited for creating rapidly activatable prodrug constructs [18, 19] was chosen to make a reversible, strongly lipophilic conjugate of SN-38 with tocopherol succinate (SN38-TS) for optimally stable incorporation into PEGylated NP, with a size adjusted to achieve high ingress efficiency and protracted retention in the target tissue [20, 21]. The specific contribution of this prodrug design to the potency and temporal pattern of the NP-mediated antiproliferative effect was examined in experiments comparing neuroblastoma cell growth inhibition by nanoencapsulated SN38-TS and its aliphatic ester isomer (isoSN38-TS) at a range of NP doses and exposure durations, with free SN-38 included as a reference. In vivo therapeutic efficacy of SN38-TS impregnated NP was next evaluated against established large tumors in a mouse xenograft model of neuroblastoma.

Materials and Methods

Synthesis of SN-38 derivatives and NP formulation

The phenolic ester conjugate SN38-TS (Fig. 1 and Supplementary Fig. S1A) was prepared by direct coupling of SN38 (98% pure, AK Scientific, Union City, CA 94587, USA) with D-α-tocopheryl hemisuccinate (>96% pure, Spectrum Chemical, New Brunswick, NJ, USA) in a mixture of 1-methylpyrrolidinone (1-MP) and dichloromethane (DCM) induced by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and catalyzed by 4-dimethylaminopyridine tosylate (DPTS). The isomeric aliphatic ester conjugate, isoSN38-TS, (Fig. 1 and Supplementary Fig. S1B) was obtained by reacting 10-Boc-SN38 prepared as described in [22], D-α-tocopheryl hemisuccinate, EDC and DPTS in DCM followed by removal of the Boc-protective group from the intermediate by treatment with trifluoroacetic acid and dimethylsulfide in DCM. The synthesis of the both derivatives is described in detail in Supplementary data.

Figure 1.

Figure 1

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Structures of SN-38 and its tocopherol succinate derivatives investigated in the present study.

Poly(D,L-lactide)-b-poly(ethylene glycol) with methoxy-terminated poly(ethylene glycol) block (HO-PLA-mPEG, 50,000:5,000, polydispersity: 1.35) was purchased from Advanced Polymer Materials (Montreal, QC, Canada). To make its covalently labeled fluorescent derivatives, HO-PLA-mPEG was first acylated with N-tert-butoxycarbonyl-6-aminohexanoic acid using EDC and DPTS as an activator and a catalyst, respectively. After removal of the Boc-protection, the resulting amino-functionalized polymer was reacted with BODIPY558/568 or BODIPY630/650-X N-succinimidyl esters. The labeled polymer conjugates were repeatedly precipitated from DCM solutions with methanol to remove unbound fluorophores. 1H NMR analysis showed that the conjugates contained 9 to 10 μmol/g of the respective, covalently bound BODIPY fluorophores.

SN-38 conjugate-loaded and blank NP were prepared using a modification of the previously reported method adapted for producing uniformly sized, sub-100 nm nanocarriers [23]. In brief, 20 mg of Pluronic F-68 and 10 mg of SN38-TS or isoSN38-TS were dissolved in 12 ml acetone. A total of 200 mg of the particle-forming diblock polymer was then dissolved in the obtained acetonic solution, and 8 ml of ethanol were added after the complete dissolution of the organic phase components. The organic solution was injected into 50 ml of water with stirring. The solvents and excess water were removed under gradually reduced pressure, and the volume was adjusted to 5.0 ml. Glucose (5% w/v) was added to adjust the tonicity, and NP were sterilized by passing them through a 0.2 μm filter membrane (Minisart, Sartorius, Bohemia, NY, USA). Blank NP were obtained as above with drug incorporation omitted. Fluorescently labeled formulations were made by substituting 40% of the polymer with respective BODIPY conjugates. NP loading was determined spectrophotometrically against a suitable calibration curve from background-corrected UV absorbances measured according to the formula: OD370 nm − (OD330 nm + OD410 nm)/2 after extracting the SN-38 derivatives in sec-butanol. NP size was measured by dynamic light scattering and expressed as intensity.

In situ measurements of NP disassembly kinetics

The in situ approach for NP disassembly analysis employed in the present studies is based on emission spectrum changes of the particle-forming polymer covalently labeled with BODIPY558/568 upon carrier disintegration described in our previous studies [24]. In the present investigations, the integrity status of NP was monitored based on the ratio of fluorescence intensities at 612 nm and 575 nm (F612/F575) after establishing their correlation in accelerated degradation experiments as follows. Proteinase K (Sigma-Aldrich, MO, USA) was added at 150 μg/ml to NP diluted 1:1000 in PBS. Samples were incubated at 37°C and their emission spectra were taken at predetermined time points. Emission spectra were measured using λex = 540 nm, and a part of each sample was passed through an aluminum oxide membrane with a 0.02 μm pore size (Anotop, Whatman Inc., NJ, USA) impermeable to intact NP. Aliquots taken before and after filtration were digested with acetonitrile and analyzed by fluorimetry (λex/λem=540nm/575nm). F612/F575 measured at each time point was plotted as a function of the respective fraction of the fluorophore dissociated from NP. The obtained inverse correlation showing linearity up to 55% of NP disassembly (Fig. 2C) was applied for directly determining the integrity status of NP incubated in FBS at 37°C (diluted 1:1000, Fig. 2D). The F612/F575 ratio of triplicate NP samples was measured without prior separation and the respective disassembly states were calculated for each time point.

Figure 2.

Figure 2

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Size distribution and in vitro disassembly analysis of NP(SN38-TS). Size distribution measured by dynamic light scattering is shown as intensity (A). Forced degradation with Proteinase K was used to obtain fluorescence spectra of intact and semi-disintegrated NP and establish the ratio of emission intensities at 612 nm and 575 nm (λex = 540 nm) as a function of the extent of NP disassembly (B and C, respectively). NP disassembly kinetics in FBS at 37°C were determined in situ based on spectral changes (D). Data in D are presented as mean ± SD.

In vitro neuroblastoma cell growth inhibition studies

Human neuroblastoma cells (IMR-32) purchased from ATCC (Manassas, VA, USA) were stably transduced with firefly luciferase and characterized as described in [25]. For growth inhibition studies, the cells were seeded on day -1 in 96-well plates at 2% confluence. DMEM supplemented with 10% FBS was used as the medium in all cell experiments. The activity of firefly luciferase employed as a cell viability marker having a short half-life of ~3 hr in mammalian cells [26] was longitudinally monitored by luminometry using D-luciferin potassium salt (PerkinElmer, Bridgeville, PA, USA) as a substrate (50 μg/ml), and the obtained results were verified by cell counting. On day 0, cells were treated with the indicated doses of free SN-38 (initially dissolved in DMSO at 0.4 mg/ml), or blank NP and NP loaded with SN38-TS or isoSN38-TS (abbreviated as NP(SN38-TS) and NP(isoSN38-TS), respectively) diluted in the culture medium. The doses were expressed as SN-38 equivalent amounts in ng per well. Blank NP were applied at doses corresponding to those of loaded particles. After treatment, the medium was carefully removed, cells were washed and their incubation continued in fresh medium. Their viability was measured at predetermined time points as above using untreated cells as a reference.

Neuroblastoma animal model experiments

Animal studies were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of the Children’s Hospital of Philadelphia. Six week-old athymic nu/nu mice were injected subcutaneously in the flank with 107 SY5Y-TrkB cells [27] suspended in 0.1 ml of Matrigel (BD Bioscience, Palo Alto, CA, USA). The tumors were allowed to reach a size of 1 cm3, and the animals were given intravenously (IV) a total of 16 doses of NP(SN38-TS) twice a week at 200 μg SN-38 per injection (n = 8). Control animals were injected IV twice a week with equivalent amounts of either saline or blank NP (n = 9 and 8, respectively) after reaching the initial tumor size of 0.2 cm3. The tumor size, body weights and blood counts of all animals were checked regularly. In efficacy studies, mice were sacrificed after reaching the tumor volume of 3 cm3. Tumor growth over time was used to calculate and compare the progression rates in the respective animal groups. Cumulative survival of animals treated with NP(SN38-TS) vs. animals administered with blank NP or saline was monitored as another endpoint.

In an additional set of experiments, fluorescently labeled NP(SN38-TS) were administered IV to mice with tumors established as above (1 cm3). Animals were sacrificed 4, 24 and 72 hr post-injection, extensively perfused with saline, and the NP and total SN-38 contents were analyzed in tumor tissue homogenates by fluorimetry and HPLC-MS/MS, respectively. For fluorimetric analysis, samples homogenized in water were extracted in two steps with acetonitrile, the organic phase was separated by adding aqueous sodium chloride (5 M), and the amounts of the BODIPY558/568-labeled polymer were measured in the extracts using λex and λem of 540 nm and 575 nm, respectively. For HPLC analysis, tissue samples were homogenized on ice in a 1:4 (v/v) mixture of methanol and water with 1% formic acid. Due to conjugate instability, SN38-TS was first quantitatively converted to SN-38 by treatment with sodium hydroxide (1 M), followed by extraction in acetonitrile. Control sample analysis confirmed complete hydrolysis of SN38-TS to SN-38 under these conditions. The assay exhibited a linear signal response up to 1000 ng of SN-38 per gram of tissue with a limit of quantitation of 10 ng/g.

Tumor samples harvested at 24 hr from animals treated with fluorescent NP(SN38-TS) and untreated control animals were also examined by fluorescence microscopy. Tissues were fixed for 1 hr in 0.5% paraformaldehyde/PBS at room temperature, then infiltrated with 30% sucrose/PBS for 1 hr at room temperature. After sucrose infusion, the tissues were embedded in O.C.T. compound, sectioned (10 μm) and coverslipped with fluorescence mounting medium containing DAPI. Fluorescent images of the tissue sections were captured at a ×100 original magnification.

Statistical analysis

The Kruskal-Wallis non-parametric ANOVA with Dunn’s post hoc test were used to compare tumor progression rates. Differences were termed significant at p<0.05.

Results

SN38-TS and its aliphatic ester isomer isoSN38-TS were obtained at 80% and 82% yields, respectively. Consistent with their comparable, strong organophilicity (LogPoctanol/water calculated as described in [28] for SN38-TS and isoSN38-TS equals 9.63 and 9.73, respectively), the two isomeric SN-38 derivatives were encapsulated with similarly high efficiencies using the nanoprecipitation approach adapted for producing sub-100 nm sized PLA-b-PEG based NP (61±2% entrapment yield corresponding to a loading of 1.22±0.04 mg/ml). The mean diameters of NP were independent of the cargo structure and equaled 75±17 nm (Fig. 2A). Notably, the substantially less organophilic unmodified SN-38 (LogPoctanol/water = 2.67), was not amenable to encapsulation using this approach due to its rapid redistribution into the external phase with subsequent precipitation upon the removal of the organic solvents.

Disassembly kinetics of NP(SN38-TS) were investigated in fetal bovine serum. For in situ NP disassembly analysis, NP were fluorescently labeled by inclusion of BODIPY558/568-PLA-b-PEG, and changes in the BODIPY558/568 fluorophore emission accompanying disintegration of NP were monitored over 15 days. A linear inverse correlation between the extent of NP disassembly and the ratio of emission intensities at 612 nm and 575 nm (F612/F575) was established in accelerated degradation experiments (Fig. 2B, C) and applied for direct measurements of prodrug-loaded NP stability in the presence of serum proteins. These studies revealed a complex disintegration pattern with an initial rapid phase between days 0–3 (24.4±0.4% disassembly) followed by slower degradation with the NP disassembly rate again increasing after day 7 (17.3±2.3% degradation over 8 days, Fig. 2D).

In the next set of experiments, the growth inhibitory effect of NP(SN38-TS) was investigated in cultured IMR-32 neuroblastoma cells in comparison to free SN-38 and NP(isoSN38-TS) as a function of dose and exposure duration. Free SN-38 and NP(SN38-TS) applied to cells for 24 hr fully inhibited cell proliferation in the studied dose range (5–25 ng SN-38/well), whereas equivalent doses of blank NP or NP(isoSN38-TS) either had no antiproliferative effect, or resulted in only marginal cell growth inhibition (Fig. 3A, B). Notably, the cumulative cell uptake of fluorescently labeled NP(SN38-TS) over the course of the 24-hr exposure was below 0.3% for all studied doses, consistent with the shielding effect of NP surface PEGylation [29]. Cell response to NP applied over a range of time periods was next examined as a model of the variable in vivo exposure of different tumor cell populations to therapeutically adequate drug levels. Whereas cell treatment with NP(SN38-TS) over 24 hr immediately initiated profound growth inhibition at all studied doses (Fig. 3A, B), the growth of cells exposed to NP for 2 hr or 10 min revealed a bell-shaped pattern with a NP dose-dependent delay in the cell number decline onset. Thus, the time points corresponding to the respective curve maxima were 1, 2 and 4 days for NP(SN38-TS) applied at doses equivalent to 25, 10 and 5 ng SN-38 per well (Fig. 3C, D and Fig. 4A). The number of cells attained before cell death started to prevail over proliferation was inversely dependent on the treatment duration, being the highest after the 10-min exposure (Fig. 3C, D).

Figure 3.

Figure 3

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In vitro growth inhibition studies in cultured neuroblasoma (IMR-32) cells. The effect of NP(SN38-TS) (25 ng/well, 24-hr exposure) is shown in comparison to ‘no treatment’ or controls applied to cells at equivalent doses (A). The effect of NP(SN38-TS) on IMR-32 cell growth measured at 7 days post-treatment (24-hr exposure) is shown as a function of drug dose in comparison to free SN-38 and NP loaded with isoSN38-TS (B). The effect of different durations of cell exposure to NP(SN38-TS) on cell growth is shown for formulation doses equivalent to 25 and 10 ng SN-38 per well (C and D, respectively). Data are presented as mean ± SD.

Figure 4.

Figure 4

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The distinct growth profiles of IMR-32 cells after a brief (10 min) exposure to NP(SN38-TS), free SN-38 and NP(isoSN38-TS) (A, B, and C, respectively) at doses corresponding to 5–25 ng SN-38 per well. Data are presented as mean ± SD.

Interestingly, after the transient growth phase observed at the shorter exposure durations, the number of viable cells measured 7 days post-treatment with NP(SN38-TS) returned to values equal to or lower than those at day 0 (i.e. <16±3 thousand cells/well) with the exception of the shortest exposure combined with the lowest examined NP dose (5 ng SN-38/well, 10 min), where the cell number at day 7 amounted to 70% of the peak value corresponding to day 4 post-treatment (134±100 thousand cells/well). The bell-shaped cell growth patterns observed after the shorter exposures to NP(SN38-TS) are in contrast to the immediate and complete growth inhibition by free SN-38 (Fig. 4B), as well as to unrestricted expansion of cells treated with NP(isoSN38-TS) (Fig. 4C) pointing to the important role of the pharmacophore regeneration rate enabled by the SN38-TS prodrug design.

After administration of NP(SN38-TS), the amounts of NP and the drug determined as total SN-38 in tumor tissue 4 hr post-treatment averaged 0.37±0.12% and 1.15±0.11% dose/g, respectively (Fig. 5A). This result, together with the drug-depleted state of NP in the blood at this time point (8.0±0.7% and 4.6±1.0% of the NP and SN-38 equivalent dose measured in the blood compartment, respectively), suggest that by 4 hr a sizeable fraction of the NP-associated drug had partitioned from the carrier, with the released compound representing a larger portion of the drug detectable in the tumor tissue. While NP continued to accumulate in the tumor over 24 hr reaching 0.79±0.09% dose/g, the total amount of SN-38 decreased to 0.86±0.10% dose/g, likely reflecting rapid elimination of the free, unprotected drug and a comparatively slow build-up of the NP-encapsulated SN38-TS prodrug. Tumors harvested at this time point revealed a primarily focal pattern of the NP distribution and a marked decrease in the number of cells in the tumor parenchyma in comparison to tumors explanted from untreated control animals (Fig. 5B, C). Mitotic activity was also strongly reduced by the treatment as evidenced by a notably lower fraction of nuclei staining positive for Ki67, a robust marker of cell proliferation [30] (Supplementary Fig. S2). The amounts of NP and drug in the tumors declined in a comparable proportion over the next 48 hr to 0.25±0.00% and 0.32±0.06% dose/g, respectively (Fig. 5A), suggesting that during the period from 24 to 72 hr post-injection the drug was predominantly in the form associated with NP and subject to the processes governing the elimination of the carrier.

Figure 5.

Figure 5

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Tumor uptake and retention of SN-38 and NP following intravenous administration of NP(SN38-TS) at a dose corresponding to 200 μg SN-38 in a mouse xenograft model of neuroblastoma. Tissue levels of the drug and the carrier were measured 4, 24 and 72 hr post-injection and expressed as % dose per gram (A). The presence of fluorescently labeled NP was confirmed in tumors harvested 24 hr post-injection by fluorescent microscopy (B) in comparison to tumor tissue samples from untreated animals included as background controls (C). Data in A are presented as mean ± SE.

NP(SN38-TS) administered twice a week caused tumor shrinkage from 1.05±0.13 to 0.28±0.09 cm3 in the course of the first week of treatment, and the tumor sizes remained stable throughout the rest of the treatment period (0.21±0.04 cm3, Fig. 6A). A 100% cumulative survival was maintained for 111 days after the last administered dose. During this period, tumor regrowth occurred in the NP(SN38-TS) treated animals at a rate of 0.011±0.002 cm3 per day, remarkably slower than that observed in control groups receiving saline or blank NP, i.e. 0.27±0.17 cm3 and 0.24±0.11 cm3 per day, respectively (p<0.05 vs. each control; no statistically significant difference in the tumor progression rate between the controls). Thus, over 7 days the tumors in the latter two groups progressed from their initial sizes of 0.20±0.10 and 0.27±0.14 cm3 to 2.12±1.28 and 1.95±0.88 cm3, respectively. This is in stark contrast to the animals pretreated with 16 doses of NP(SN38-TS), in which an increase in the average tumor size from 0.21±0.04 to 1.51±0.74 cm3 occurred over 16 weeks. The observed decrease in the rate of tumor regrowth was paralleled by a histopathological shift from immature neuroblastoma to a ganglioneuroma-like appearance of the tumors harvested from animals sacrificed 18 weeks after cessation of the NP(SN38-TS) treatment (Supplementary Fig. S3).

Figure 6.

Figure 6

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Therapeutic efficacy of NP(SN38-TS) in animals with established large tumors (1 cm3) in a mouse xenograft model of neuroblastoma. Mice were administered 16 doses equivalent to 200 μg SN-38 per injection twice a week and sacrificed after reaching the tumor size of 3 cm3 (n = 8). The tumor growth and animal survival (A and B, respectively) were monitored over the course of treatment and after treatment cessation. The tumor size progression is shown for NP(SN38-TS) treated animals until the removal of the first animal reaching the threshold tumor volume of 3 cm3 (111 days after the last administered dose). Event-free survival is shown in comparison with control animal groups (blank NP and saline). Data are presented as mean ± SD.

Discussion

The development of clinically viable nanomedicine-based cancer treatment strategies poses considerable challenges in constructing formulations with scalable and effective designs and optimizing their therapeutic performance and safety [5, 10, 31]. In addition to the biocompatibility and eliminability of their components, NP designed for cancer pharmacotherapy should ideally combine a small and uniform size, which is a prerequisite for extravasation and retention in the tumor tissue [20], with stable entrapment of the NP-encapsulated drug to prevent its activity loss and minimizing off-target effects [10]. However, a reduction in the carrier size to the desirable sub-100 nm range and the resultant increase in the surface area/volume ratio can disrupt NP-drug association during the formulation process [32, 33] or cause rapid drug escape after in vivo administration [7, 9].

In the present study, this challenge is met by combining the use of a formulation method providing sub-100 nm injectable NP made of biodegradable and bioeliminable components with a reversible chemical modification of the drug molecule, creating a strongly hydrophobic prodrug optimized for NP-based delivery and applied here to a camptothecin analog, SN-38. Despite its proven anticancer potential, the clinical use of SN-38 is hampered by its low solubility in water or pharmaceutically acceptable vehicles [34], rapid conversion to its pharmacologically inactive form in vivo, and severe side effects [35]. Attempts have been made to develop formulation approaches enabling parenteral administration of solubilized SN-38 in its chemically unmodified form. Complexation with cyclodextrins has recently been reported to dramatically increase solubility of SN-38 (from 0.08 μg to 50 μg per mL) [36]. However, despite these improvements, this value remains orders of magnitude lower than that achievable by applying chemical modifications to the parent molecule. Thus, in the clinic the drug is administered as its water soluble precursor, irinotecan. SN-38 regeneration from irinotecan requires enzymatic cleavage, which takes place primarily in the liver [16, 37]. This process is inefficient in comparison to other pathways contributing to the elimination of irinotecan [38], resulting in low but variable bioactivation rates, typically not exceeding 3–4% [39]. The pattern of subsequent tissue distribution of the regenerated SN-38 is non-specific, and its fraction eventually reaching the tumor in its functional form is further diminished by extensive metabolism that SN-38 undergoes in the liver and plasma [38]. In this context, its NP-based delivery as SN38-TS offers important, therapeutically relevant advantages: i) a significant fraction of the drug reaches its site of action in association with the carrier and remains protected by entrapment in the NP matrix-forming polymer; ii) upon release from the particle, the rapid drug recovery characteristic of phenolic ester prodrug derivatives [19] effectively outcompetes concurrent inactivation processes, iii) the enzyme-independent activation mechanism enables drug regeneration directly in tumor tissue and allows its protracted retention at effective local levels as suggested by the results of our in vivo studies.

The essential role of the prodrug design was elucidated in the series of in vitro experiments showing effective NP(SN38-TS)-mediated cell growth inhibition directly dependent on the exposure time and the dose of the nanoencapsulated prodrug, whereas cell treatment with NP loaded with its slowly activatable isomer uniformly resulted in a marginal antiproliferative effect. These in vitro experiments also prompt two important observations regarding the mechanism of the cell growth inhibitory effect mediated by NP(SN38-TS). First, these NP were effective at inducing cell death at all exposure durations, despite the negligibly low NP internalization. Second, the kinetic pattern of cell growth inhibition was distinct from that of free SN-38 when the exposure was limited to 2 hr or 10 min. In contrast to the immediate onset of the antiproliferative effect mediated by the free drug, the number of viable cells continued to increase for a certain period of time after the transient exposure to NP(SN38-TS), suggesting that in the latter case DNA damage accumulated gradually until the repair capacity of a cell was exceeded leading to the initiation of the apoptosis pathway. Based on these results, the response to NP treatment may be expected to occur on a protracted time scale with a lag inversely dependent on the drug accessibility and varying within a broad range for sets of cells experiencing different levels of drug exposure within the tumor tissue.

The potent antiproliferative effect of NP(SN38-TS) observed in the absence of cell uptake or extensive carrier disassembly within all studied exposure periods is noteworthy, suggesting that upon direct contact with cells a fraction of the therapeutic payload sufficient for providing therapeutically effective intracellular drug levels could be released by structurally intact particles. Rapid transfer of strongly lipophilic molecules from carriers interacting directly with cell membranes has been demonstrated previously in several studies [40–42]. The high capacity of membrane lipids for tocopherol derivatives (“tocophilicity”) [43] is consistent with this mechanism underlying the results of our experiments. Interestingly, while direct physical interaction between NP and cancer cell membranes is an explicitly in vitro phenomenon [2], the existence of an analogous process driving the rapid early redistribution of a fraction of the therapeutic payload in vivo is prompted by the partially drug-depleted state of NP in the blood concomitant with intratumoral drug accumulation exceeding that of NP at 4 hr post-injection in our experiments. Similar to our in vitro observations, this early phase of drug release in vivo may not be attributed to NP disintegration based on the distinct time scales of these processes. These findings indicate that at early time points, in addition to SN-38 directly delivered in the prodrug form by NP, the contribution of alternative pathways may be significant. These likely involve a one-step drug transfer from NP to tumor endothelial cells and exposed extracellular matrix elements or a multistep process where part of the therapeutic payload partitioning from the carrier is accommodated, transported and deposited by circulating cells. Notably, at later time points the intratumoral drug levels closely paralleled those of NP suggesting that at 24 and 72 hr post-administration the majority of the drug in the tumor was in the carrier-associated, protected form, and its fate was governed primarily by that of the tumor-localized particles.

The regression and sustained inhibition of tumor regrowth mediated by NP(SN38-TS) suggest that therapeutically effective local levels of the drug were achieved and maintained using NP-based delivery. Interestingly, the tumor regrowth rate strongly reduced in animals pretreated with NP(SN38-TS) compared to control groups suggests that NP, while not eradicating the tumor completely, could achieve drug exposure levels sufficient to exert phenotypic selection by providing a survival advantage to quiescent cells. This is in agreement with the cell cycle-specific mode of action of camptothecins targeting the DNA replication phase and predominantly affecting actively dividing cells, as opposed to other families of chemotherapeutics, such as topoisomerase II inhibitors, whose cytotoxicity is considerably less dependent on active replication [44]. This mechanism, complementary to the direct cell toxic effects of the nanoparticulate drug, is also supported by the markedly reduced mitotic activity 24 hr post-treatment and is presumably responsible in part for the sustained antitumoral effect observed after cessation of NP(SN38-TS) treatment accompanied by a favorable change in tumor cell phenotype. Notably, because of the inability to administer SN-38 as a free compound in the sufficient dose due to the solubility issues mentioned above, the “free drug” control group could not be included in our therapeutic efficacy studies. However, others have shown that intravenous administration of its water soluble precursor, irinotecan, at a dose comparable to that used in our study was unable to provide long-term survival in a mouse neuroblastoma xenograft model [45].

In the prodrug-loaded NP formulation evaluated in these studies, the role of TS released concomitantly with SN-38 upon prodrug activation may extend beyond its passive contribution as a hydrophobizing promoiety assisting in formulation of stably loaded sub-100 nm NP. Several studies have shown that through mitochondria-dependent signaling [46] TS exerts therapeutically relevant effects in vitro and in animal models of several types of cancer [47, 48]. The incorporation of SN-38 and TS in nanocarriers in the form of a common molecular precursor, SN38-TS, allows both compounds to take advantage of the NP-based delivery, biodistribution and intratumoral retention. Although the extent of the pharmacological contribution of TS to the observed anticancer activity of NP(SN38-TS) cannot be directly addressed through experiments, the recovery of TS in the tumor tissue synchronized and synlocalized with that of SN-38 could potentially play a role by potentiating the effect of the camptothecin drug. This is in agreement with previous investigations demonstrating strongly enhanced antineoplastic potency of chemotherapeutic agents when used in combination with TS [48]. Interestingly, both selective induction of apoptosis in cancer cells, including highly malignant neuroblastoma cells [49], and promotion of differentiation in several malignant cell types [50, 51] have been reported as therapeutically relevant effects exerted by TS. The ability of TS to cause differentiation or enhance the effect of other cell differentiating agents [52] may potentially have contributed to the aforementioned histopathological change observed in animals pretreated with NP(SN38-TS).

Conclusions

This study demonstrates feasibility and therapeutic efficacy of a drug delivery approach based on the use of sub-100 nm biodegradable nanocarriers and rational prodrug design applied to SN-38, a camptothecin drug with proven anticancer potential currently not in clinical use due to significant pharmacological and formulation challenges. In the nanoencapsulated form, its rapidly activatable precursor, SN38-TS, was shown to provide a strong and sustained therapeutic effect in a mouse xenograft model of neuroblastoma, as evidenced by rapid tumor regression, extended inhibition of tumor regrowth and prolonged animal survival (median survival of 182 days as a result of 16 doses of treatment). Our results point to a complex mechanism of intratumoral delivery, likely involving relatively rapid uptake of the active compound partially redistributed from NP and its gradual accumulation in the NP-bound form, the latter exhibiting more protracted retention in the tumor tissue. The strong anticancer effect of the prodrug-loaded NP and the markedly less aggressive behavior exhibited subsequently by the tumors are both highly relevant in the therapeutic context of neuroblastoma treatment. Our findings point to the importance of identifying and addressing critical formulation challenges for increasing effectiveness and improving translational potential of experimental nanomedicine-based approaches as new cancer treatment modalities.

Supplementary Material

supplement

Figure S1. 1H NMR spectra of 1 (SN38-TS) and 3 (isoSN38-TS) shown in A and B, respectively.

Figure S2. The early effect of NP(SN38-TS) treatment on tumor tissue histology and cell proliferation. NP(SN38-TS) were administered intravenously to mice bearing neuroblastoma xenografts at a dose corresponding to 200 μg SN-38. Tumors were harvested 24 hr post-treatment. Tumors from untreated animals were included as controls. A notable decrease in the overall number of cells and in mitotic activity was observed in NP-treated tumor samples immunostained with antibodies to Ki67 (B vs. A) as follows. Paraformaldehyde-fixed, paraffin-embedded tumors were sectioned at 7 μm. Slides deparaffinized with xylene and gradually rehydrated through a series of ethanol dilutions into PBS were subjected to antigen retrieval in citrate buffer for 5 minutes. Tissue was blocked in 10% goat serum/PBS for 30 minutes at room temperature and incubated with a rabbit monoclonal antibody to Ki67 (GeneTex) diluted 1:50 in 1% BSA/PBS overnight at 4°C. After washing, the tissues were incubated with goat anti-rabbit AlexaFluor 488 (Invitrogen Molecular Probes) secondary antibody diluted 1:200 in 1% BSA/PBS for 1 hr at room temperature. Tissues coverslipped with Vectashield fluorescence mounting medium containing DAPI were photographed at an original magnification ×200.

Figure S3. NP treatment-induced histomorphological differentiation of tumor cells in Hematoxylin-Eosin stained tissue samples. In comparison to tumors explanted from untreated (control) mice, tumors harvested from NP(SN38-TS)-treated animals 18 weeks after treatment cessation exhibit a shift to a ganglioneuroma-like appearance based on the comparative evaluation of the histological differentiation parameters (enlargement of nuclei, the presence of nucleoli, development of increasing amounts of cytoplasm). This is in contrast to the histological pattern of the control tumor samples revealing densely packed, undifferentiated cells with reduced amounts of cytoplasm. Original magnification ×200.

Acknowledgments

The authors thank Dr. Ganesh Moorthy and Praveen Srivastava (The Children’s Hospital of Philadelphia) for assisting with tissue sample analysis. This research was supported in part by the National Institutes of Health grants, R01-HL111118 (MC) and R01-CA094194 (GMB), Alex’s Lemonade Stand Foundation, the V Foundation, the Audrey E. Evans Endowed Chair (GMB), and The Children’s Hospital of Philadelphia Research Funds including the William J. Rashkind Endowment, Erin’s Fund, and The Kibel Foundation (RJL).

Footnotes

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Associated Data

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Supplementary Materials

supplement

Figure S1. 1H NMR spectra of 1 (SN38-TS) and 3 (isoSN38-TS) shown in A and B, respectively.

Figure S2. The early effect of NP(SN38-TS) treatment on tumor tissue histology and cell proliferation. NP(SN38-TS) were administered intravenously to mice bearing neuroblastoma xenografts at a dose corresponding to 200 μg SN-38. Tumors were harvested 24 hr post-treatment. Tumors from untreated animals were included as controls. A notable decrease in the overall number of cells and in mitotic activity was observed in NP-treated tumor samples immunostained with antibodies to Ki67 (B vs. A) as follows. Paraformaldehyde-fixed, paraffin-embedded tumors were sectioned at 7 μm. Slides deparaffinized with xylene and gradually rehydrated through a series of ethanol dilutions into PBS were subjected to antigen retrieval in citrate buffer for 5 minutes. Tissue was blocked in 10% goat serum/PBS for 30 minutes at room temperature and incubated with a rabbit monoclonal antibody to Ki67 (GeneTex) diluted 1:50 in 1% BSA/PBS overnight at 4°C. After washing, the tissues were incubated with goat anti-rabbit AlexaFluor 488 (Invitrogen Molecular Probes) secondary antibody diluted 1:200 in 1% BSA/PBS for 1 hr at room temperature. Tissues coverslipped with Vectashield fluorescence mounting medium containing DAPI were photographed at an original magnification ×200.

Figure S3. NP treatment-induced histomorphological differentiation of tumor cells in Hematoxylin-Eosin stained tissue samples. In comparison to tumors explanted from untreated (control) mice, tumors harvested from NP(SN38-TS)-treated animals 18 weeks after treatment cessation exhibit a shift to a ganglioneuroma-like appearance based on the comparative evaluation of the histological differentiation parameters (enlargement of nuclei, the presence of nucleoli, development of increasing amounts of cytoplasm). This is in contrast to the histological pattern of the control tumor samples revealing densely packed, undifferentiated cells with reduced amounts of cytoplasm. Original magnification ×200.

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