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Published in final edited form as: Int J Radiat Oncol Biol Phys. 2020 Nov 27;109(5):1483–1494. doi: 10.1016/j.ijrobp.2020.11.052

Localized Delivery of Cisplatin to Cervical Cancer Improves Its Therapeutic Efficacy and Minimizes Its Side-Effect Profile

Cinzia Federico 1,†, Jennifer Sun 1,2,†, Barbara Muz 1, Kinan Alhallak 1,2, Pippa F Cosper 1,3, Naoshad Muhammad 1, Amanda Jeske 1,2, Amanda Hinger 1, Stephanie Markovina 1,4, Perry Grigsby 1,4, Julie K Schwarz 1,4, Abdel Kareem Azab 1,2,4,*
PMCID: PMC8592040  NIHMSID: NIHMS1733854  PMID: 33253820

Abstract

Purpose:

Cervical cancer represents the fourth most frequent malignancy in the world among women, and mortality has remained stable for the past four decades. Intravenous cisplatin with concurrent radiation therapy is the standard-of-care for patients with local and regional cervical cancer. However, cisplatin induces serious dose-limiting systemic toxicities and recurrence frequently occurs. In this study, we aimed to develop an intra-cervical drug delivery system that allows cisplatin release directly into the tumor and minimize systemic side effects.

Methods and materials:

Twenty patient biopsies and five cell lines treated with cisplatin were analyzed for platinum content using inductively coupled plasma mass spectrometry. Polymeric implants loaded with cisplatin were developed and evaluated for degradation and drug release. The effect of local or systemic cisplatin delivery on drug biodistribution as well as tumor burden were evaluated in vivo, in combination with radiation therapy.

Results:

Platinum levels in patient biopsies were 6-fold lower than the levels needed for efficacy and radiosensitization in vitro. Cisplatin local delivery implant remarkably improved drug specificity to the tumor, and significantly decreased accumulation in the blood, kidney, and other distant normal organs, compared to traditional systemic delivery. The localized treatment further resulted in complete inhibition of tumor growth.

Conclusions:

The current standard-of-care systemic administration of cisplatin provides a sub-therapeutic dose. We developed a polymeric drug delivery system that delvered high doses of cisplatin directly into the cervical tumor, while lowering drug accumulation and consequent side effects in normal tissues. Moving forward, these data will be used as the basis of a future first-in-human clinical trial to test the efficacy of localized cisplatin as adjuvant or neoadjuvant chemotherapy in local and regional cervical cancer.

Keywords: drug delivery, cervical cancer, cisplatin, systemic toxicity, radiation therapy

INTRODUCTION

Cervical cancer represents the fourth most frequent malignancy in the world among women, with estimations of 570,000 new cases and more than 300,000 deaths annually.1,2 Despite encouraging improvements in screening and prevention, cervical cancer mortality has remained stable for the past 4 decades.3 Additionally, 90% of deaths occur in low- and middle-income countries, which lack widespread implementation of cervical cancer screening and prevention programs.4

Current treatment options for cervical cancer include radical hysterectomy or definitive chemoradiation depending primarily on the clinical stage of the disease.5 The current standard-of-care radiation therapy for locally advanced cervical cancer consists of conventionally fractionated radiation to the pelvis (once daily) in addition to hypofractionated boost dose delivered directly to the tumor via intracavitary brachytherapy. Systemic cisplatin-based chemotherapy is concurrently administered with radiation, which has been shown to improve local control as well as overall survival outcomes compared to radiation alone in randomized controlled clinical trials.6,7

Cisplatin (Cis-Pt) is an alkylating antineoplastic agent8 used to treat several malignancies including cervical cancer.9–12 Despite Cis-Pt’s potent anticancer activity, it possesses serious dose-limiting side effects such as nausea and vomiting, kidney damage, neuropathy, hearing loss, and bone marrow suppression.13–15 These dose-limiting side effects can negatively impact clinical efficacy of Cis-Pt, causing chemotherapy noncompliance and discontinuation as potential drivers for inefficacious therapy and disease relapse.16 The current standard-of-care Cis-Pt dosage for cervical cancer treatment is once weekly intravenous (IV) injection of 40–70 mg/m2; however, previous studies have shown that 24 hours after IV injection of a 100 mg/m2 dose resulted in intra-tumoral platinum (Pt) concentration of only 0.9 ng/mg.17 This concentration is far below the effective Pt amount needed in cervical cancer cells to achieve cytotoxic effects in vitro, as demonstrated by previous reports.18 Therefore, the development of a new drug delivery system designed to maximize the intra-tumor accumulation of Cis-Pt while minimizing the dose-limiting side effects has the potential to dramatically improve therapeutic outcomes.

In this study, we developed an easy to implement polyethylene glycol (PEG)-based drug delivery implant with fast dissolution properties for localized delivery of Cis-Pt chemotherapy to cervical cancer. We hypothesize that these implants will improve Pt accumulation in the cervical tumor and improve therapeutic efficacy in combination with radiation treatment, while simultaneously reducing Pt distribution in other organs and Pt associated side effects.

MATERIALS AND METHODS

Chemicals and reagents

Poly(ethylene glycol)-3350 (PEG3350) and poly(ethylene glycol)-400 (PEG400), cis-Diammineplatinum(II) dichloride (Cis-Pt), trypan blue, and nitric acid (HNO3) were purchased from Sigma Aldrich (St Louis, MO). The near infrared (NIR) lipophilic tracer DiR was purchased from Invitrogen (Eugene, OR). Corning Matrigel Basement Membrane Matrix Growth Factor Reduced (Corning, New York, NY) was prepared following manufacturer instructions.

Cell culture

Human cervical cancer cell lines C33A, Me-180, HT3, Caski, and SiHa were purchased from American Type Culture Collection (ATCC; Manassas, VA). Cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; Gibco Life Technologies), 2 mmol/L of L-glutamine, 100 μg/mL penicillin, and 100 μg/mL streptomycin (Corning CellGro, Mediatech, Manassas, VA). All cells were cultured at 37°C, 5% CO2 in a NuAire water jacket incubator (Plymouth, MN).

Cervical cancer patient biopsies

Patients with biopsy-proven cervical carcinoma were enrolled into an Institutional Review Board (IRB)-approved prospective study in which cervical tumor biopsies were obtained 3 weeks into chemoradiation treatment. Informed consent was obtained from all patients with an approval from the Washington University in St. Louis Institutional Review Board Committee and in accord with the Decla-ration of Helsinki (protocol # 201105374). All patients were treated with definitive chemoradiation treatment, which consisted of external beam radiotherapy to the pelvis (50.4 Gy) four days per week using intensity modulated radiation therapy with a split-pelvis technique with six weekly brachytherapy boosts to a dose of 6.5 Gy per fraction to point A, starting at week 1. Concurrent with radiotherapy, all patients received the conventional treatment of cisplatin (40 mg/m2, intravenous (IV)) weekly. The cervical tumor sample was collected immediately prior to delivery of the third brachytherapy treatment, approximately half-way through the treatment course. Samples were collected, immediately snap frozen, and processed for storage in the Siteman Cancer Center Tissue Bank at Washington University in St. Louis. The average days between cisplatin administration and biopsy was 4.5 days (range 1 – 17 days).

Pt accumulation in cervical cancer human biopsies

To determine tissue Cis-Pt accumulation within the patient biopsy samples, we performed tissue digestion and inductively coupled plasma mass spectrometry (ICP-MS) to quantify the levels of Pt in the samples, as previously described.19,20 Briefly, biopsy samples were thawed and precisely weighed (±0.1mg) before being digested in concentrated nitric acid (HNO3) overnight. Samples were then diluted with deionized water to a final 5% HNO3 (v/v), and digested using a microwave digestion system (MARS 6 Microwave Digestion System, CEM, Matthews, NC) at 200°C for 45 minutes. Samples were analyzed by ICP-MS (ELAN DRC II ICP-MS, Perkin Elmer, Inc., USA) for Pt content, against a calibration curve of Pt standards of 0, 0.1, 1, 10, 50,100 and 250 parts per billion (ppb) in 5% HNO3, which were prepared from a 10 parts per million Pt standard solution (Inorganic Ventures, Christiansburg, VA). Terbium was used as an internal standard throughout the ICP-MS analysis. The average Pt content in clinical biopsies (Av-Pt-Clin) was calculated.

In vitro efficacy of Cis-Pt

The in vitro cytotoxicity on cervical cancer cell lines was assessed using MTT assay (Sigma Aldrich) as previously described.21 Briefly, cells were seeded at 5 × 103 cells/well in 96-well plates overnight at 37°C to promote their adhesion to the plates. They were treated with increasing concentrations of Cis-Pt of 1, 2.5, 5, and 10 μM. After 48 hours of treatment, MTT solution was added to the cells for 3 hours, then the stop solution was added to dissolve the formazan crystals overnight. The wells were read with SpectraMax i3 multi-mode microplate spectrophotometer (Molecular Devices, San Jose, CA) at 570nm. Dose-response curves were graphed and the average dose-response curve was calculated. The average half-maximal inhibitory concentration (Av-IC50) of Cis-Pt from all five cell line was also calculated.

Pt content in cell lines in vitro

Cervical cancer cell lines (10×106 cells) were treated Cis-Pt at Av-IC50 for 48 hours. Cells were then lifted with trypsin (0.05% Trypsin-EDTA, Gibco Life Technologies), spun down, and washed three times, to eliminate free Cis-Pt. Cell pellets were then weighed, digested and analyzed by ICP-MS, and the average Pt content in cell lines in vitro (Av-Pt-in vitro) was calculated.

In vitro radio-sensitization with Cis-Pt

Clinically relevant Pt concentration (Clin-conc) was estimated as a fraction of Av-IC50 based on the relative difference between tissue Pt contents in clinical biopsies and in vitro: Av-IC50 * (Av-Pt-Clin /Av-Pt-in vitro). The radiosensitizing effect of Cis-Pt on cervical cancer cell lines was assessed using MTT assay. Briefly, cells were treated with or without Cis-Pt at Av-IC50 (6μM) or Clin-conc (1μM) for duration of 24 or 48 hours. Additionally, cells were also treated with or without a single fraction dose of 6 Gy (4 Gy/min) using an RS2000 160kV X-ray Irradiator equipped with a 0.3 mm copper filter (Rad Source Technologies Inc, Buford, GA), at the beginning of the treatment. At the end of each treatment, MTT assay was performed to determine survival of the cells.

The combination effect between Cis-Pt treatment and radiation was evaluated by calculating the combination index (CI) using the formula: CI = (Ea x Eb)/Eab, where Ea and Eb are the individual effects (fractional killing; 0≤Ei≤1) and Eab is the combination effect of drugs a and b. This index represents the ratio between the predicted combination effect and the actual combination effect. When CI =1, the effect is additive. When CI < 1, the effect is supra-additive; when CI > 1, the effect is sub-additive.

In vitro clonogenic survival

C33A cervical cancer cells (0.5×106 cells/condition) were plated in T25 flasks 24 hours before treatment. Cells were treated with Cis-Pt at 0, 1, 3, or 6μM for 4 hours, and subjected to irradiation at 0 or 6 Gy (4 Gy/min). Cells were then lifted with trypsin, counted, and were plated in triplicates for each condition at 500 cells per well in 24 well plates. After 12 days of culture, cells were fixed and stained with 0.5% Crystal Violet (Sigma Aldrich) in methanol, washed, and dried. The number of clones resulted in each well were analyzed by ImageJ software (NIH, Bethesda, MD).

PEG-implant preparation

PEG-implants were prepared from PEG3350 and PEG400 mixtures with mass ratio of 80:20. The PEG-implants were also loaded with trypan blue (for in vitro dissolution studies), near infrared (NIR) dye DiR (for in vivo dissolution studies), or Cis-Pt (for in vitro release, in vivo biodistribution and efficacy studies). The different loads were homogenously stirred into the molten polymers mixture, before cooling.

Cis-Pt PEG-implants for in vivo biodistribution and efficacy studies were prepared at 2 mg Cis-Pt/g PEG and allowed to cool in molds. Implants for in vivo studies were obtained by biopsy aspiration needles (BD, Vernon Hills, IL) and weighed approximately 10±0.5 mg each and contained 0.02 mg of Cis-Pt. Administration of each implant is equivalent to a dose of 1 mg/kg in a 20g animal.

In vitro dissolution and drug release of PEG-implant

Trypan blue implants were placed into a 6-well plate and allowed to dissolve in PBS (3 ml/well) over a shaker at 37°C. Dissolution rate of the implant was monitored by photographs taken at multiple time points (0–30 min) until disappearance. The size of the remaining implant at each time point was analyzed using ImageJ software.

To evaluate the release profile of Cis-Pt from implants in vitro, Cis-Pt (10mg /g PEG) was loaded to the implants. Implants were placed into a 6-well plate PBS (3 ml/well) over a shaker at 37°C. Release rate was monitored by sampling 100 μL of buffer at different time points (0–30 min) and replacing with fresh buffer. The amount of released Pt was measured by ICP-MS. The release was calculated as: (Amount at time point – Blank) / (Total amount – Blank) × 100%.

In vivo dissolution rate

Approval for the animal studies was obtained from the Ethical Committee for Animal Experiments. Balb/c mice, female, 6- to 8-week-old (Charles Rivers Laboratories, Wilmington, MD) were anesthetized with ketamine/xylazine. To evaluate the Cis-Pt release profile from the implants in vivo, a small incision was made to the abdominal wall and a DiR-loaded implant was inserted into the peritoneal cavity of each mouse (n=3). Release rate was monitored by imaging the infrared signal in each mouse at different time points (0–30 min) using Pearl NIR fluorescent imager (LI-COR Biotechnology, Lincoln, NE). The in vivo release was calculated as: (Area with infrared)/ (Area of the whole abdomen) × 100%.

In vivo biodistribution

C33A cells (2×106/mouse) were suspended in 50% Matrigel- 50% DMEM media, and injected subcutaneously around the lower back of 10 athymic nude mice, female, 6- to 8-week-old (Charles River Laboratories). Tumor progression was confirmed after 3 weeks as tumor volume of >200 mm3, measured by caliper and calculated using the formula: V = 0.5 × a × b2, in which a and b are the major and minor axis of the tumor, respectively. Mice were randomized into 2 groups (n=5) and treated with: (1) tail vein injection of free Cis-Pt at the dose of 1 mg/kg (Cis-Pt-IV), or (2) intra-tumoral implantation of Cis-Pt-loaded implant at the dose of 1 mg Cis-Pt/kg (Cis-Pt-Implant).

To mimic the anatomic structure of the cervix for local delivery, an os-like cavity was developed in the center of the subcutaneous tumor by taking out a biopsy from the center of the tumor using a Jamshidi bone marrow biopsy aspiration needle (BD, Vernon Hills, IL). For the local delivery, implants were inserted into the tumor cavity by a biopsy needle. The cavity was then closed using VetClose surgical glue (Henry Schein, Dublin, OH). Mice were sacrificed 24 hours post-treatment and various organs (tumor, heart, kidney, liver, spleen and blood) were harvested, weighed, digested with nitric acid/microwave, and analyzed for Pt content using ICP-MS. The concentration of Pt in each tissue was determined as: Pt Amount / Tissue Weight.

In vivo tumor penetration

Subcutaneous cervical tumors were inoculated in 6 athymic nude mice as described above. Mice were treated with intra-tumoral empty implant (n=3), or intra-tumoral Cis-Pt-loaded implant (n=3). After 24 hours, mice were sacrificed and the tumors were resected. The tumors were fixed with PFA, cut to half, one half for ICP-MS elemental analysis, and one half for immunofluorescence.

For ICP-MS, tumors were washed with PBS and cut into three layers, inner, middle, and outer, corresponding to the distance from drug implant (approximately 1mm each layer), digested, and analyzed as discussed previously. Pt level is represented as ng Pt / g tissue and adjusted for empty implant background signal.

For immunofluorescence, tumor were frozen in OCT compound (Fisher Scientific, Pittsburgh, PA), and sectioned to 10 μM slices using Leica CM1950 Cryostat (Leica, Wetzlar, Germany). Cryosections were blocked with 10% FBS, stained with 1:200 dilution of Cisplatin-modified DNA antibody (CP9/19) (Novus Biologicals, Centennial, CO) for 1 hour at room temperature, washed with PBS, followed by staining with 1:1000 dilution of goat anti-Rat IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Invitrogen, Carlsbad, CA) for 30 minutes at room temperature, and washed with PBS. Sections were counter-stained with ProLong Gold Antifade Mountant with DAPI (Invitrogen) and analyzed by AxioPlan 2 Fluorescence Microscope (Carl Zeiss, Oberkochen, Germany).

In vivo efficacy

C33A cells (2×106/mouse) were suspended in 50% Matrigel- 50% DMEM media, and injected subcutaneously around the lower back of 42 athymic nude mice, female, 6- to 8-week-old. Tumor progression was confirmed after 3 weeks, and mice were then randomized into 6 groups (n=7) and treated with: (1) tail vein injection of PBS (Vehicle-IV), (2) Cis-Pt-IV at 1 mg/kg, (3) Cis-Pt-Implant at 1 mg Cis-Pt/kg, (4) Vehicle-IV + radiation (5 days x 2 Gy/ day), (5) Cis-Pt-IV + radiation, and (6) Cis-Pt-Implant + radiation.

In a separate experiment, we compared the effect of fractionated dose to a single high-dose. C33A cells (2×106/mouse) were suspended in 50% Matrigel- 50% DMEM media, and injected subcutaneously around the lower back of 14 athymic nude mice, female, 6- to 8-week-old (Charles Rivers Laboratories). Tumor progression was confirmed after 3 weeks, mice were then implanted with Cis-Pt-loaded implant, local implantation (1 mg/kg) and randomized into 2 groups (n=7) and treated with: (1) fractionated radiation (5 days x 2 Gy/ day), mimicking external beam radiation; or (2) a single high dose of 8Gy, mimicking brachytherapy. Tumor progression was monitored by measurement of tumor sizes in all treatment groups every two days.

Tumor progression was monitored by measurement of tumor sizes in all treatment groups every two days. Radiation treatment for mice were 2 Gy/day for 5 consecutive days, or a single high dose of 8 Gy, using an RS2000 160kV X-ray Irradiator equipped with a 0.3 mm copper filter (Rad Source Technologies Inc, Buford, GA), starting immediately after the first Vehicle or Cis-Pt treatment. The mice were protected with lead shielding such that only the subcutaneous tumors were exposed to radiation.

Statistical analysis

All experiments were performed in at least triplicates, and cell line experiments were repeated at least three times. Results were expressed as means ± standard deviation, and statistical significance was analyzed using Student’s t-test and ANOVA. P values less than 0.05 were used to indicate statistically significant differences.

RESULTS

The effect of Cis-Pt as a radio-sensitizer in cervical cancer

We first established the cytotoxicity profiles of Cis-Pt in human cervical cancer by creating dose-response curves for five cervical cancer cell lines (C33A, Me-180, HT3, Caski, and SiHa). We found the average dose-response curve and further calculated the average IC50 (Av-IC50) using this curve (Fig. S1). The Av-IC50 was found to be 6 μM and represents the in vitro efficacious concentration.

Furthermore, we determined the Pt content in 20 tumor biopsies from cervical cancer patients approximately three weeks into definitive treatment with chemoradiation, and compared this to the Pt content found in cell lines treated with Cis-Pt at AV-IC50. Patient characteristics are presented in Table 1 and Pt content in biopsies was represented as a function of time since the last cisplatin injection, showing that the time of biopsy was not a major factor for the level of Pt within the tumor (Fig. S2). Pt levels in cell lines treated at in vitro efficacious concentration of 6 μM averaged to be 2560 ng Pt/g tissue, while Pt levels detected in patient tissues averaged to be only 430 ng Pt/g tissue (Fig. 1A). Collectively, these results demonstrate that drug accumulation in the patient tissues was about 6-fold lower, which would be equivalent to the result of a 1 μM treatment condition. According to the does-response curves found previously, this clinically relevant concentration of 1 μM would have limited cytotoxicity.

Table 1.

Clinical Characteristics of Patients with Cervical Tumor Biopsies

Clinical Characteristic All patients (n=20)
Median Age 53.7 years (range 33–76)
FIGO stage Value (%)
 IB1 1 (5)
 IB2 5 (25)
 IIB 3 (15)
 IIIA 1 (5)
 IIIB 9 (45)
 IVB 1 (5)
Histology
 Squamous 17 (85)
 Adenocarcinoma 3 (15)
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Abbreviations: FIGO, International Federation of Gynecology and Obstetrics, 2009. staging.

Figure 1. The effect of intracellular cisplatin (Cis-Pt) on anti-tumor efficacy.

Figure 1.

Figure 1.

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(A) Platinum (Pt) concentration in patient biopsy samples (n=20) and in cell lines treated at Av-IC50 (n=5), measured by inductively coupled plasma mass spectrometry (ICP-MS). (B) Relatively number of surviving clones as % of untreated. P-values represent comparisons against 0μM Cis-Pt condition in respective radiation dose (** p < 0.01).

Next, we tested how low Cis-Pt accumulation in cervical cancer tissues would affect Cis-Pt as a radio-sensitizer. We treated cell lines with Cis-Pt at the in vitro efficacious concentration of 6 μM or at the clinically relevant concentration of 1 μM, with or without concurrent radiation. We found that 6 μM Cis-Pt was able to induce significant sensitization to radiotherapy in all cell lines (Fig. S3A); on the other hand, 1 μM Cis-Pt induced limited radio-sensitization in the cell lines (Fig. S3B). Furthermore, the combination index (CI) was calculated to compare the predicted combination effect and the actual combination effect of Cis-Pt and radiation in both cases. Specifically, CI for 6 μM treatment showed supra-additive effect, whereas CI for 1 μM treatment showed sub-additive effect (Table S1).

Next we sought to test how this discrepancy in Cis-Pt levels affect the clonogenic ability in cervical cancer cells. C33A cell lines were treated with 0, 1, 3, or 6 μM Cis-Pt, with or without concurrent radiation. Analyzing the relative number of surviving clones revealed that 1μM concentration was unable to limit clonogenic cell survival of cervical cancer cell lines, neither alone nor in combination with radiation. In contrast, higher doses (3 and 6 μM) significantly restricted clonogenic growth (Fig. 1B).

These results imply that the synergistic interaction between Cis-Pt and irradiation can be only achieved with a higher Pt accumulation in tumor tissues, which can potentially be accomplished with localized delivery of Cis-Pt into the tumor, prior to radiation therapy.

Development of localized fast release delivery system for Cis-Pt

We used a combination of polyethylene glycol (PEG) with low and high molecular weights, PEG400 and PEG3350, for the development of a fast release delivery implant. We characterized the degradation of the implant in vitro, and found that it degraded gradually within 25 minutes (Fig. 2A). Similarly, Cis-Pt loaded implants released its content gradually into the buffer and Pt content reached a plateau around 25 minutes (Fig. 2B). Furthermore, comparable results were observed in vivo; DiR-loaded implants inserted into the peritoneal cavity in mice degraded and released the dye within 25 minutes, as shown in representative NiR images (Fig. 2C) and quantitative measurements (Fig. 2D).

Figure 2. Characterization of polyethylene glycol (PEG) implants.

Figure 2.

Figure 2.

Figure 2.

Figure 2.

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(A) In vitro dissolution profile of empty implants, calculated as percent of original size at 0 min. (B) In vitro drug release profile of Cis-Pt loaded implants, calculated as % of total dose. (C) Representative images for in vivo dissolution of DiR-loaded implants in mice (n=5). (D) In vivo dissolution profile, calculated as percent area in the abdomen with DiR signal.

In vivo biodistribution of Cis-Pt after localized or systemic administration

Patients with cervical cancer undergo brachytherapy treatment as a standard part of care. During each of the treatments, a device is implanted through the cervix tumor and into the uterus, presenting us with an opportunity to introduce local drug delivery in conjunction with this implant. We therefore developed a subcutaneous model of cervical cancer tumor in which we created a cavity in the center to mimic the anatomical structure of the human cervix, allowing insertion of the drug delivery devices (Fig. 3A). We then administered mice with systemic Cis-Pt (Cis-Pt-IV) according to clinical practice, or with localized Cis-Pt implants (Cis-Pt-Implant). The biodistribution profiles for the two treatment groups were compared by measuring the Pt amount in various organs 24 hours after IV or local implantation. We found that Pt accumulated in the tumor was 73.4-fold higher in local delivery (3896 ng Pt/g tissue) compared to systemic delivery (53 ng Pt/g tissue). On the contrary, Pt levels detected in blood and kidney were significantly lower in local implant compared to systemic delivery (Fig. 3B and C). Additionally, tumor-to-blood ratio was calculated to be 80.3 for localized delivery and 0.3 for systemic delivery (Fig. 3C).

Figure 3. In vivo localized Cis-Pt delivery compared to systemic Cis-Pt delivery.

Figure 3.

Figure 3.

Figure 3.

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(A) Schematic for in vivo subcutaneous tumor model and the local delivery of the device. (B) In vivo biodistribution profile of Cis-Pt (n=5), measured by Pt levels in various organs, 24 hours after Cis-Pt-Implant or Cis-Pt-IV administration. (* p < 0.05; ** p < 0.01; *** p < 0.001). Insert on top right shows a zoomed in view of the biodistribution profile. (C) Ratios for Pt levels in tumor, blood, and kidney, as well as tumor/blood Pt ratios seen in Cis-Pt-Implant and Cis-Pt-IV groups.

In vivo tumor penetration of Cis-Pt implants

We next asked how far Cis-Pt would be able to diffuse from the implants into the depth of the tumor. We directly assessed Pt distribution in the tumors using two different methods: quantitatively using ICP-MS, and qualitatively using immunofluorescence (IF) from each tumor (Fig. 4A). ICP-MS analysis revealed Cis-Pt penetrates all layers of the tumor. The levels of Cis-Pt was inversely correlated with the distance from the implant; the tumor layers closest to the implant had high concentration of Pt, and outer layers had moderate levels of Pt, all of which are higher than what we detected in tumor biopsies from patients (Fig. 4B). For IF, we stained for Cis-Pt-DNA adducts and imaged regions at the center or the edge of tumor sections. We saw that Cis-Pt-DNA adducts were abundant in the center region of the tumors and to a lower extent in the edges of the tumor (Figure 4C).

Figure 4. In vivo tumor penetration of Cis-Pt released from implants.

Figure 4.

Figure 4.

Figure 4.

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(A) Assessment of Pt distribution in different sections of the tumors after treatment with empty or Cis-Pt implants using two different methods: ICP-MS and immunofluorescence (IF). (B) Pt concentration in each tumor layer from Cis-Pt implant group (n=3) by ICP-MS analysis, adjusted with background signal from empty implant group (n=3). (C) Fluorescence microscopy images of regions of tumor sections, stained with antibody for Cis-Pt induced DNA adducts (AF488) and counterstained with DAPI. Gray lines indicate the edge of the tissue.

In vivo efficacy of localized Cis-Pt delivery

Lastly, we compared the effect of Cis-Pt on cervical tumor progression when delivered locally or systemically, with or without concurrent radiation. We found that systemic delivery of Cis-Pt did not alter tumor progression of cervical tumors in vivo, while localized delivery of the same dose resulted in a complete inhibition of tumor growth (Fig. 5A and Fig. S4A). Furthermore, when Cis-Pt treatments were combined with fractionated radiation (2 Gy x 5 days, representing clinical dose), we found that systemic Cis-Pt delivery was able to improve treatment outcome compared to radiation alone, while localized Cis-Pt treatment profoundly improved effect of radiation in vivo (Fig. 5B and Fig. S4B), implying augmented radio-sensitization.

Figure 5. In vivo efficacy of Cis-Pt implants for cervical cancer.

Figure 5.

Figure 5.

Figure 5.

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(A) Tumor progression for treatment conditions Vehicle-IV, Cis-Pt-IV, or Cis-Pt-Implant, without irradiation. (B) Tumor progression for treatment conditions Vehicle-IV, Cis-Pt-IV, or Cis-Pt-Implant, with 2 Gy/day irradiation for 5 consecutive days. (C) Tumor progression for Cis-Pt-Implant with fractioned radiation (2 Gy/day x 5 days) or high dose brachytherapy (8 Gy once). (* p < 0.05; ** p < 0.01; *** p < 0.001).

In a separate experiment, we investigated whether a single high dose of radiation (8 Gy; similar to the brachytherapy boost portion of standard-of-care radiotherapy for cervix) would cause a different outcome compared to 5 consecutive fractionated doses (2 Gy/day; representing conventionally fractionated external beam irradiation). We found that the localized Cis-Pt implant showed better results when combined with single high dose compared to combination with fractionated therapy (Fig. 5C and Fig. S4C), suggesting that administration of localized Cis-Pt immediately prior to brachytherapy may result in considerable clinical benefit.

DISCUSSION

Intravenous Cis-Pt with concurrent radiation therapy is the gold standard treatment for patients with local and regional cervical cancer. While this treatment is effective for many patients, recurrence of disease occurs in 30–60% of the patients.5,7,22–24 Additionally, this treatment regimen is associated with serious dose-limiting toxicities that may result in sub-therapeutic drug concentrations.15,25 In our study, we demonstrated that the levels of Cis-Pt found in biopsies isolated from cervical cancer patients (treated with standard-of-care 40 mg/m2) were significantly lower than the levels of Pt found in cell lines after treatment with an efficacious concentration in vitro (Av-IC50). These low levels of Pt did not achieve significant killing or radio-sensitivity in vitro (using both clonogenic assay and MTT proliferation assay) and in vivo. This suggests that the current clinical standard-of-care systemic administration of Cis-Pt provides a sub-therapeutic dose for the treatment of cervical cancer. Achieving higher intra-tumoral doses may be needed to promote better therapeutic efficacy, but most certainly will result in significant toxicities if administered systemically. Therefore, there is an urgent need for a drug delivery system to specifically deliver high doses of Cis-Pt directly to the tumor, while avoiding Cis-Pt accumulation and consequent side effects in normal tissues.

Previous studies have shown that systemic delivery systems for chemotherapy (such as nanoparticles) showed promising results for treatment of metastatic cervical cancer due to the disseminated nature of the disease.26 However, more than 80% of cervical cancer cases are diagnosed at local or regional stages.27 Therefore, a systemic approach may not be the ideal strategy for the treatment of local and regional cervix cancer cases. It has been demonstrated that localized delivery of chemotherapy in various solid tumors improve therapeutic efficacy in the tumor and limit systemic toxicities.28–34 Similarly, a localized treatment approach in cervical cancer has a great potential for improving local tumor control and reducing side effects. In particular, the cervix is accessible for intra-vaginal implantation of local drug delivery devices directly adjacent to the cancer tissue. Current intra-vaginal devices for delivery of chemotherapy in cervical cancer include vaginal rings, vaginal gels, and cervical patches.35–39 These devices are placed on the external side of the cervix which in many cases does not promote delivery into the deeper parts of the cervical tumor. Moreover, these have long-term release properties, which often become their main limitation due to clearance by the physiological discharge of mucus produced by glands of the cervix,40 which hinder their effectiveness.

In this study, we developed a drug delivery system which can be implanted into the cervical cavity and allow Cis-Pt release into the depth of the tumor tissue. Moreover, it was designed to promote fast-release of Cis-Pt to circumvent clearance by the physiological secretions of the vaginal tract. Since most cervical cancers at diagnosis are local and regional, there are potential intervention points where localized therapy could supplement existing treatments, and the current standard-of-care brachytherapy treatments would provide an ideal time to locally administer Cis-Pt if fast drug release can be achieved. This device can be implanted into the cervix and release its load within the timeframe of the radiotherapy treatment and act as radio-sensitizing adjuvant/neoadjuvant therapy.

We successfully developed an implant based on PEG, an FDA approved biocompatible polymer used for drug delivery and tissue engineering applications., and is highly soluble in an aqueous milieu, such as the cervix.41–43 The implant is solid at room temperature to allow insertion into the depth of the cervical cavity. We found that the implant’s dissolution and drug release rates were suitable for a fast release application, as it was able to dissolve and release its cargo within 30 minutes in vitro and in vivo.

Choosing an in vivo animal model was challenging. Cervical cancer animal models are extremely limited.44 Orthotopic models, while it represents the biological and anatomical characteristics of cervical cancer, their use in the context of intra-cervical drug delivery is a challenge due to the small size of the mouse cervix, and the corresponding technical challenges in developing implants of this size. Alternatively, subcutaneous models provide the proper size of the tumor, but lack the anatomical structure needed for intra-tumoral implantation. Therefore, we developed a mouse model with subcutaneous cervical cancer tumor, where we created a cervix-like cavity within the center of the tumor, allowing localized delivery using the PEG-implant. This model was chosen for the in vivo experiments including biodistribution and efficacy.

Biodistribution studies revealed clear advantages for the localized implant comparing to IV injection of the same dose of Cis-Pt. The localized implant demonstrated a drastic 73.4-fold higher accumulation of Cis-Pt in the tumor compared to systemic delivery. Additionally, it resulted in 3.7-fold and 5.26-fold lower accumulation in the blood and kidney, and negligible levels in distant normal organs. Another measurement of specificity and relative safety of a drug treatment is the tumor-to-blood ratio; we found that the localized Cis-Pt delivery achieved an impressive 80-fold higher drug content at the tumor site compared to the peripheral blood, whereas systemic delivery exhibited an opposite tumor-to-blood ratio of 0.3. This demonstrates remarkable improvement of the specificity of Cis-Pt delivery to the tumor. Moreover, in vivo systemic Cis-Pt was not able to induce a therapeutic effect alone or in combination with radiotherapy. On the contrary, localized delivery of Cis-Pt resulted in complete inhibition of tumor growth, alone or in combination with fractionated radiotherapy or brachytherapy. In summary, this study demonstrated that localized intra-cervical Cis-Pt delivery dramatically improved therapeutic outcomes of Cis-Pt-based chemo-radiotherapy in cervical cancer. Similarly, this kind of localized approach can be utilized for delivery of other potent drugs that are clinically limited by high systemic toxicity, such as DNA damage response inhibitors.

CONCLUSIONS

In conclusion, we have successfully created a polymeric delivery implant for fast and local Cis-Pt delivery to cervical cancer, which yielded outstanding biodistribution profile with superior tumor drug accumulation and significantly lower drug accumulation in normal tissues compared to systemic delivery. It further achieved an enhanced in vivo anti-tumor therapeutic efficacy. Moving forward, the data of this study will be used as the basis of a future first-in-human clinical trial to test the efficacy of localized Cis-Pt as adjuvant/neoadjuvant chemotherapy in local and regional cervical cancer.

Supplementary Material

1

Figure S1. Determining IC50 in cervical cancer cell lines. Dose response curves of cervical cancer cell lines treated at 0, 1, 2.5, 5, and 10μM Cis-Pt for 48 hours. The average dose response is shown in red and the average IC50 (Av-IC50) is calculated to be 6 μM.

Figure S2. Levels of Pt in patient biopsies as a function of days from Cis-Pt administration to biopsy.

Figure S3. Radiosensitization effect of Cis-Pt in 5 individual cervical cell lines. (A) Radiosensitization effect of Cis-Pt at Av-IC50 (6 μM) with single dose 6 Gy radiation. (B) Radiosensitization effect of Cis-Pt at 1 μM with single dose 6 Gy radiation.

Figure S4. In vivo efficacy of Cis-Pt implants for cervical cancer. (A-C) Absolute tumor volumes (* p < 0.05; ** p < 0.01; *** p < 0.001).

Acknowledgments

Funding: The study was partially supported by an award from the National Institutes of Health (NIH) and the National Cancer Institute of the NIH (U54CA199092). J.S. was supported by the Spencer T. and Ann W. Olin Fellowship for Women in Graduate Study at the Washington University in St. Louis. K.A was supported by the National Center for Advancing Translational Sciences of the NIH under Award Number TL1TR002344.

Footnotes

Conflict of interest: C.F., J.S., J.K.S. and A.K.A. have a patent pending regarding the technology described in this paper.

Data sharing statement: Research data are stored in an institutional repository and will be shared upon request to the corresponding author.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1

Figure S1. Determining IC50 in cervical cancer cell lines. Dose response curves of cervical cancer cell lines treated at 0, 1, 2.5, 5, and 10μM Cis-Pt for 48 hours. The average dose response is shown in red and the average IC50 (Av-IC50) is calculated to be 6 μM.

Figure S2. Levels of Pt in patient biopsies as a function of days from Cis-Pt administration to biopsy.

Figure S3. Radiosensitization effect of Cis-Pt in 5 individual cervical cell lines. (A) Radiosensitization effect of Cis-Pt at Av-IC50 (6 μM) with single dose 6 Gy radiation. (B) Radiosensitization effect of Cis-Pt at 1 μM with single dose 6 Gy radiation.

Figure S4. In vivo efficacy of Cis-Pt implants for cervical cancer. (A-C) Absolute tumor volumes (* p < 0.05; ** p < 0.01; *** p < 0.001).

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