Doxorubicin-Loaded Polymeric Meshes Prevent Local Recurrence After Sarcoma Resection While Avoiding Cardiotoxicity

Abstract

Surgery is the only potentially curative treatment for localized soft tissue sarcomas. However, for sarcomas arising in the retroperitoneum, locoregional recurrence rates are 35-59% despite resection. Doxorubicin (DOX) is the standard first-line systemic chemotherapy for advanced soft tissue sarcoma, yet its intravenous administration yields limited clinical efficacy and results in dose-limiting cardiotoxicity. We report the fabrication and optimization of a novel electrospun poly(caprolactone) (PCL) surgical mesh coated with layers of a hydrophobic polymer (poly(glycerol monostearate-co-caprolactone), PGC-C18), which delivers DOX directly to the operative bed following sarcoma resection. In xenograft models of liposarcoma and chondrosarcoma, doxorubicin-loaded meshes (DoM) increased overall survival four-fold compared to systemically administered DOX and prevented local recurrence in all but one animal. Importantly, mice implanted with DoMs exhibited preserved cardiac function, while mice receiving an equivalent dose systemically displayed a 23% decrease from baseline in both cardiac output and ejection fraction 20-days post administration. Collectively, this work demonstrates a feasible therapeutic approach to simultaneously prevent post-surgical tumor recurrence and minimize cardiotoxicity in soft tissue sarcoma.

Teaser

Doxorubicin-loaded meshes prevent recurrence and cardiotoxicity in murine resection models of liposarcoma and chondrosarcoma.

Significance/Layman language

A proof-of-principle study in animal models shows that a novel local drug delivery approach can prevent tumor recurrence as well as drug-related adverse events following surgical resection of soft tissue sarcomas.

Graphical Abstract

Introduction

Soft tissue sarcomas are a heterogeneous group of localized mesenchymal tumors that comprise roughly 1% and 6-8% of adult and pediatric malignancies, respectively(1). Approximately 15% of all sarcomas arise in the retroperitoneum(2,3). Despite complete (R0 or R1) resection, locoregional recurrence rates for patients with retroperitoneal sarcomas (RPS) are 35-59% even in experienced hands(4,5), and the 5-year overall survival (OS) rates are 50-69%(6-8). Due to size, complex growth, and anatomical restrictions, obtaining clean margins for soft tissue sarcoma is particularly difficult. Further, there is no universal clinical definition of a negative margin, and local recurrence occurs even in cases designated R0 (microscopically negative margin) by pathological examination after surgery(9,10). Death and morbidity amongst patients with retroperitoneal sarcoma are often from sequelae of local recurrence, which may occur even if a resection is classified as R0. Neoadjuvant radiotherapy showed no improvement in abdominal recurrence-free survival compared to surgery alone in a recent phase III randomized trial(11). Currently available adjuvant systemic chemotherapy demonstrates limited efficacy for retroperitoneal soft tissue sarcomas(12,13). Thus, locoregional control represents an unmet need for many patients, including after R0/R1 resection.

Doxorubicin (DOX) is the standard first-line systemic chemotherapy for most advanced sarcomas. Unfortunately, systemic delivery of DOX yields a low overall response rate (14-21% in liposarcoma), attributable to poor drug delivery and dose-limiting side effects(13). Due to its short half-life (8 minutes), achieving efficacious DOX levels in tumor tissue requires a large bolus dose, which is limited by off-target cardiotoxicity(14). Patients who develop doxorubicin-associated cardiomyopathy suffer a 50% one-year mortality(14). Protocols for slow, continuous infusion avoid bolus dosing, which contributes to off-target toxicity, but anti-tumor activity declines under these protocols(15). The ideal solution for using DOX after complete (R0/R1) resection would allow for focused, efficacious drug delivery to resection sites where residual disease remains undiscovered, with little drug dissemination to off-target sites.

Biomaterial drug delivery systems (i.e., nanoparticles, hydrogels, wafers) are under investigation for targeting and/or providing sustained drug delivery in various cancer types(3). Pegylated liposomal doxorubicin (Doxil^®) significantly prolongs the DOX half-life to ~90 hours (16). However, a phase II randomized trial of Doxil versus doxorubicin for advanced or metastatic soft tissue sarcoma showed only a 10% response rate for Doxil, statistically similar to doxorubicin(17). Several groups report polymeric implants to deliver chemotherapeutics over a period of weeks or months(18-23). These implants typically encapsulate hydrophobic small molecule drugs into polymeric depots to achieve desired release kinetics(24,25). However, delivery of DOX over several weeks or months is challenging due to its hydrophilic nature, and parameters influencing release kinetics from polymeric drug delivery devices remain incompletely defined. Doxorubicin delivery via a hydrogel exhibits some efficacy in pre-clinical models of osteosarcoma after intra-tumoral injection. However, the hydrogel delivery system induces systemic toxicity, and it is unclear if the hydrogels could be used in the post-tumor resection setting or scaled to the necessary size needed for larger tumors, such as retroperitoneal sarcomas(26,27). To overcome the shortcomings associated with pre-existing local DOX delivery systems, we established a set of design requirements: 1) be surgically implantable, 2) utilize established and scalable methods of manufacturing, 3) be composed of safe, non-toxic, and biodegradable materials, 4) retain drug efficacy after encapsulation, 5) demonstrate sustained release for at least 4 weeks with minimal burst release (defined as less than 10% of cargo over the first 3 days), 6) prevent local disease recurrence and improve survival, and 7) avoid cardiotoxicity while maintaining efficacy.

In this work, we report the design and optimization of novel free base DOX-loaded meshes (DoMs). DoMs afford tunable release rates via degradation over several weeks to months depending on composition. By incorporating DOX in its hydrophobic, free base form, into a novel polymeric mesh, DoMs avoid substantial burst release seen in continuous infusion protocols(28) and drug-loaded hydrogels and wafers(29). This in turn prevents acute toxicity and affords prolonged local drug delivery at efficacious concentrations. We describe the fabrication and efficacy of DoMs for implantation in sarcoma resection beds, explore the physical and pharmacokinetic properties of the meshes, and identify key structural and material features required for optimal activity. Then, we demonstrate antitumor efficacy, advantageous biodistribution, and abrogation of the cardiotoxic effects of DOX treatment in two murine xenograft models of human sarcoma.

Materials and Methods

Fabrication of Polycaprolactone Nanofiber Meshes

Polycaprolactone was dissolved overnight in a 30:70 mixture of dimethylformamide (DMF) and chloroform in a 20 mL glass scintillation vial at 27% w/v with or without free base DOX at 25 mg/ml. This solution was mixed thoroughly and added to a glass syringe fixed to a syringe pump. The solution was forced through an 18-gauge needle at 0.9 ml/h towards a cylindrical metal target spinning at 60 rpm across a 22.5 kV electric potential.

Coating of Nanofiber Meshes with DOX

Films were coated with 50 μL of polymer/drug solution containing either 500 mg or 250 mg of DOX and 10% w/v poly(glycerol monostearate-co-caprolactone), PGC-C18. The solution is cast onto the film via syringe, applying even coatings to each side, drying for 1 minute between each layer.

In Vitro Release Profile of DOX

Sink conditions for release experiments consisted of phosphate buffered saline (PBS) supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. A volume of 10 mL was used for meshes. At each time point, 5 mL was taken for analysis and replaced with 5 mL of fresh release buffer, ensuring greater than 10-fold solubility excess. Meshes were sealed in scintillation vials and incubated on a shaker at 37 °C (n = 3 per group). Aliquots were withdrawn at days 0, 1, 2, 3, 4, 7, 14, 21, and 28, and DOX fluorescence was measured on a Biotek Synergy plate reader.

Cell Culture

The CS1 cell line was established from a resected high grade human CHSA tumor and propagated in vitro(30). LP6 liposarcoma tumor and cells was obtained from patient immediately after resection (IRB-approved protocol 05-434). All cell lines are tested on a biweekly basis for mycoplasma by PCR via SouthernBiotech’s mycoplasma detection kit (Cat. No. 13100-01).

In Vitro Assessment of Cytotoxicity

LP6 and CS1 cells were plated in 12-well plates at a seeding density of 5 x 10⁴ cells/well in RPMI supplemented with 10% (v/v) FBS, 100 U/mL penicillin, 100 μg/ml streptomycin and grown overnight at 37 °C in a humidified 5% CO₂ incubator. The plated cells were then washed with PBS before exposure to the meshes via a transwell system. After 24 hours, we removed the meshes, and the cells were incubated with the DOX released from the meshes for an additional 2 days. Cell viability was then assessed via MTS assay and normalized to an untreated control. In between co-incubation, meshes were kept in sink conditions at 37 °C in a humidified 5% CO₂ incubator.

In Vivo Locoregional Recurrence Model and Film Implantation

All animal experiments were approved and conducted in accordance with the guidelines for humane care and use of laboratory animals from the Institutional Animal Care and Use Committee of the Dana-Farber Cancer Institute (Boston, MA) and of Massachusetts General Hospital (Boston, MA). CS-1 chondrosarcoma tumors were established by injection of 2x10⁶ CS-1 cells into the upper dorsum of 6-8 weeks old female Nu/J mice (Jackson Laboratories, Bar Harbor, ME). LP6 tumors were established by subcutaneous implantation of 2x2x2-mm tissue fragments (Dana-Farber Cancer Center, Boston, MA) at the upper dorsum of 6-8 week-old female Nu/J mice. We confirm that we obtained written informed consent from the patients, that the studies were conducted in accordance with recognized ethical guidelines (e.g., Declaration of Helsinki, CIOMS, Belmont Report, U.S. Common Rule), and that the studies were approved by an institutional review board (Dana-Farber Cancer Center, IRB protocol 05-434). A power analysis indicated 8 animals per group. When primary tumors reached a volume of >500 mm³, an R1 (macroscopically complete with microscopically positive margin) resection was performed under isoflurane anesthesia whereby all gross visible tumor tissue was removed. The incision was closed with wound clips. Tumor recurrence was monitored by daily inspection and palpation. Locoregional recurrence was defined as any palpable subcutaneous nodule and recorded as either in-field (underneath the film) or out-of-field (outside the film area). Animals were randomized to receive one of the treatment groups. Films were implanted over the resection bed and secured at the corners with 5-0 silk suture. The incision was closed with wound clips. Body weight was measured every other day for the first week after surgery. Time to recurrence and survival was assessed up to 100 days after surgery. Mice were euthanized when tumors reached 2-cm, body weight dropped 15% within one week, or upon significant clinical deterioration.

Cardiotoxicity Studies and Echocardiography

Cardiotoxicity studies were performed in non-tumor bearing Nu/J mice. The DOX films were implanted at the upper dorsum as described previously. Echocardiography was performed on days 5 and 20 following treatment, and measures of cardiac function were recorded. Mice were anesthetized with 3% isoflurane, which was reduced to 1.5% isoflurane during echocardiography. Images were collected using a 14.0-MHz linear probe (Vivid 7; GE Medical System, Milwaukee, WI). Body temperature was maintained at 37°C during echocardiography using a warming lamp. The fractional shortening, ejection fraction, cardiac output, end-diastolic volume (EDV), end-systolic volume (ESV), heart rate and left ventricle (LV) mass were calculated on an EchoPAC workstation (GE Healthcare, Wauwatosa, WI). Animals were closely monitored for weight loss or clinical signs of deterioration and were euthanized accordingly per animal facility protocol.

Statistical Analysis

Three or more experimental replicates from each experiment were compiled. Data represent mean ± SD, with n values listed in figure captions or legends where appropriate. GraphPad Prism was used to plot all bar and line graphs and perform statistical analysis. Statistical analysis performed includes unpaired T test and log-rank (Mantel-Cox) test as indicated in figure captions. For all data, p < 0.05 was considered significant.

Data and materials availability:

All reported data are available in the main text or the supplemental materials. Additional raw data and information is readily available upon request.

Results

Hydrophobic meshes exhibit ideal structural and chemical properties for extended release of DOX

Finely tuning the drug delivery release kinetics is a crucial design challenge for successful clinical translation as it dictates the exposure of tumor and healthy tissue to cytotoxic drugs. Because non-drug loaded polymeric meshes are already safely and routinely used in many surgeries, they represent an ideal platform to deliver high drug payloads while mitigating drug concentration in healthy tissues (Fig. 1). However, to successfully deploy this novel drug delivery platform for DOX delivery, we must optimize its mechanical and pharmacokinetic properties. Thus, we explored three aspects of mesh architecture: 1) hydrophobic coatings, 2) buttress degradation rates, and 3) drug loading location (loaded within polymeric fibers, within hydrophobic coatings, or both).

Fig. 1. Conceptual design and use case for drug-loaded polymeric meshes.

(A) Diagram of drug-loaded polymeric film post-resection of a retroperitoneal liposarcoma, (B) photo of sutured polymeric mesh in vivo.

To explore these parameters, we constructed a library of DOX-loaded meshes (DoMs) using combinations of mesh buttresses and hydrophobic polymer coatings. We fabricated buttresses from either poly(caprolactone) (PCL) nanofibers or poly(glycolic acid) (PGA) microfibers that emulate commercially available buttresses(31-33). Poly(glycerol monostearate-co-ε-caprolactone) (PGC-C18) is utilized as the hydrophobic coating. Fig. 2A depicts each component of the mesh architectures. We loaded free base DOX within the PCL nanofibers during the electrospinning process, in the PGC-C18 coating, or a combination of both. Fig. 2B shows a schematic non-drug loaded mesh with PCL nanofibers and two layers of PGC-C18 coating. The PCL buttresses were electrospun at a low pump rate to obtain nanofibers (diameter 184 ± 14 nm, n = 3 meshes, Fig. 2C). A single (Fig. 2D) or double coating (Fig. 2E) of PGC-C18 acts as a stabilizing layer to slow diffusion of water into the mesh and drug release into adjacent tissue. The PGA buttresses were electrospun at a higher pump rate to obtain thick fibers (diameter 5740 ± 153 nm, n = 3 meshes, Fig. 2F; see supplemental methods), and they were similarly coated with PGC-C18 (Fig. 2G). The surface of PCL or PGA meshes are hydrophobic, as determined by contact angle measurements (contact angles 118° and 132°, respectively Fig. S1A). Tensile testing revealed that the polymer base impacted the Young’s modulus of the DoMs as the PGA microfiber meshes were stiffer than the PCL nanofiber meshes. However, the external PGC-C18 coatings had relatively minimal effect on stiffness. Specifically, the PGA and PCL meshes without coatings exhibited a Young’s modulus of 49 and 13 MPa respectively, while the meshes with coatings showed a Young’s modulus of 57 and 8 MPa respectively (fig. S1B).

Fig. 2. Characterization of mesh architectures.

(A) Key for mesh naming convention, (B) schematic of mesh construction. Scanning electron microscopy (SEM) images of (C) PCL nanofibers (PCL-0-X), (D) PCL nanofibers with 1 layer of PGC-C18 (PCL-1-X), (E) PCL nanofibers with two layers of PGC-C18 (PCL-2-X), (F) PGA electrospun buttress (PGA-0-X), (G) PGA electrospun buttress coated with PGC-C18 (PGA-1-X).

DOX release kinetics are finely tunable by adjusting mesh components

To obtain the optimal mesh architecture for DOX release, we evaluated a small library of meshes for differences in drug release over 28 days. Fig. 3 and Table S1 describe the mesh performance. First, we measured DOX released from meshes with a PGA buttress and 0, 1, or 2 layers of PGC-C18 over top of a DOX-loaded layer of PGC-C18 (Fig. 3A). Increasing layers of PGC-C18 on the mesh afforded slower and more uniform release profiles (Fig. 3B,C). We analyzed drug release kinetics with the Korsmeyer-Peppas equation to quantitatively compare release constants. For 0, 1, and 2 layers, K-values were 0.25 ± 0.12, 0.14 ± 0.02, 0.1 ± 0.01, respectively (Fig. S2A-C). Next, we evaluated the effect of the buttress material using meshes with two layers of PGC-C18 over top of a DOX-loaded PGC-C18 layer and either a PCL or PGA buttress (Fig. 3D). The PCL buttresses showed decreased burst release and less variability between meshes when compared to PGA meshes (Fig. 3E,F). We attribute the decreased release rate and greater consistency to slower degradation of the polymer base. By day 21, the PGA meshes lost structural integrity, while the PCL meshes remained intact throughout the duration of the study. K-values for the PGA and PCL buttresses were 0.21 ± 0.09 and 0.06 ± 0.01, respectively (Fig. S2D,E). Overall, PGA meshes demonstrated irregularities in individual release profiles, resulting in more variable K-values. Notably, the abnormal release of a single PGA-2-C mesh (K = 0.31) resulted in significantly different K-values for two independent trials of three biological replicates of PGA-2-C mesh release (Fig. S2C K = 0.1 ± 0.01, Fig. S2D K = 0.21 ± 0.09). Finally, we assessed the influence of DOX loading location by fabricating PCL meshes with DOX loaded in the PGC-C18 coating, the fibers, or both (Fig. 3G). The hybrid mesh displayed a slight burst release (8.7% of cargo released in 3 days) and a linear release after day 3 (R² = 0.98) (Fig. 3H,I). The K-values were 0.07 ± 0.01, 0.05 ± 0.001, and 0.02 ± 0.002 for the PCL-2-C, PCL-2-C/F, and PCL-2-F meshes, respectively (Fig. S2F-H).

Fig. 3. Drug release characteristics of doxorubicin-loaded meshes.

(A) Schematic representation of PGA-2-C, PGA-1-C, and PGA-0-C meshes, (B) cumulative release profile (C) doxorubicin released per day, (D) schematic representation of PGA-2-C and PCL-2-C meshes, (E) cumulative release profile, (F) doxorubicin daily release, (G) schematic representation of PCL-2-C, PCL-2-C/F, and PCL-2-F meshes, (H) cumulative release profile, (I) doxorubicin daily release. Each data point represents the average of 3 biological replicates (n = 3), and error bars represent standard deviation.

Mesh architectures influence cell cytotoxicity in vitro against CS1 and LP6 sarcoma cells

Next, we determined if DOX released from the various DOMs retained cytotoxic activity. We first confirmed that DOX is cytotoxic to liposarcoma (LP6) and chondrosarcoma (CS1) cells in vitro (CS1 IC50: DOX HCL 115 ng/mL, free base DOX 208 ng/mL; LP6 IC50: Dox HCL 267 ng/mL, free base DOX 297 ng/mL) (Fig. 4A). Employing meshes that exhibited a range of release profiles from 21% to 92% release over 28 days (Fig. S3A,B), we performed in vitro cytotoxicity assays over a period of 6-8 weeks (Fig. 4B). Unloaded meshes were not cytotoxic to CS1 and LP6 cells (Fig. S4A,B). As in the release kinetics experiments, PGA meshes lost mechanical integrity by day 21, while the PCL meshes remained intact throughout the study. For both LP6 and CS1 cells, the PCL-2-C/F design demonstrated prolonged cytotoxicity, achieving potent activity for 6 weeks against LP6 cells and 8 weeks against CS1 cells. In contrast, PGA-1-C, PGA-2-C, PCL-1-F, and PCL-0-F exhibited greater than 50% cytotoxic activity for 2-5 weeks before losing efficacy (Fig 4C,D). Due to its favorable release profile and sustained cytotoxic activity, we selected the PCL-2-C/F mesh for evaluation in vivo. Hereon, DoM will refer to the PCL-2-C/F mesh unless otherwise specified.

Fig. 4. In vitro cytotoxicity characteristics of various mesh architectures.

(A) Doxorubicin cytotoxicity against CS1 and LP6 cells, (B) schematic of the treatment protocol to assess cytotoxicity against CS1 and LP6 cells using a library of meshes, (C) cytotoxicity of the meshes against LP6 cells in culture over 8 weeks, (D) cytotoxicity of the meshes against CS1 cells in culture over 6 weeks. Each data point represents the average of 3 biological replicates (n = 3), and error bars represent standard deviation.

DoMs reduce recurrence and improve overall survival in murine models of resected chondrosarcoma and liposarcoma

We evaluated the efficacy of locally-delivered DOX via DoMs to prevent recurrence of sarcoma after resection in vivo using two established cancer models: an aggressive chondrosarcoma cell line (CS1)(34) and a patient-derived liposarcoma xenograft (LP6)(35). We established tumors on the dorsum of Nu/J mice via injection of CS-1 cells or implantation of LP6 tumor fragments. Once the tumors grew to a volume of at least 500 mm³, we performed a complete resection (i.e., removal of all visible disease, classified as R1 because margins were not evaluated by microscopy), and mice were treated according to their randomly assigned group (Fig. 5A). The total dose of DOX in the DoMs (500 μg) was approximately equivalent to the total dose of DOX given intraperitoneally, either as a single bolus dose or weekly doses (20 mg/kg).

Fig. 5. In vivo efficacy evaluation of the PCL-hybrid dox-loaded mesh.

Schematic of the murine sarcoma resection model (A), Kaplan-Meyer curves for the CS1 recurrence (B) and overall survival (C), Kaplan-Meyer curves for the LP6 recurrence (D) and overall survival (E). ****p < 0.0001, log-rank test, DoM-treated group vs. surgery alone.

Amongst mice bearing CS-1 tumors, none treated with the DoM developed local recurrence. In contrast, animals in the control groups (surgery alone, intraperitoneal DOX as single dose or weekly dose, and unloaded film treatment) showed median recurrence of 11, 11, 10, and 12.5 days, respectively (Fig. 5B). DoM-treated animals exhibited improved overall survival with a median OS of 93 days compared to 23, 18.5, 27.5, and 26 days for the other treatment groups, respectively (Fig. 5C). In the LP6 tumor bearing mice, only one animal treated with the DoM (500ug) recurred locoregionally and died within the 100-day window. Conversely, only one animal in the untreated control group survived to 100 days. Median time to recurrence for the surgery alone, intraperitoneal DOX as single dose or weekly dose, and unloaded films was 9.5, 11, 10, and 11 days (Fig. 5D), with corresponding median survivals of 18.5, 22, 21, and 19.5 days, respectively (Fig. 5E). Overall, only 3% of mice in control groups survived during the 100-day window for both CS1 and LP6, whereas 89% and 44% of the DoM treated mice survived in LP6 and CS1 tumor bearing mice, respectively, with sacrifice required for extensive, detectable metastatic disease. Body weight did not decrease in either sarcoma model with the DoM-treated mice (Fig. S5A,B). DoM-treated mice exhibited a statistically significant longer time to recurrence (Fig. S5C,D) and overall survival (Fig. S5E,F) compared to all other groups.

DoMs efficiently deliver DOX to local tissue while preventing accumulation in healthy tissue

Next, we performed a biodistribution study to characterize DOX accumulation at local and distant tissues in non-tumor bearing mice receiving either free DOX (intraperitoneal injection, 20 mg/kg) or surgically implanted DoM (500 μg, approximately 20 mg/kg). At days 1, 5, and 30, local tissue (site of film implantation in the upper dorsum) and all major organs were collected, individually homogenized, and then assessed for DOX concentration via HPLC. On day 1 post-treatment, DOX concentration in DoM-treated mice exceeded 3,000 ng/g in local tissue whereas local DOX accumulation was only 133 ng/g in animals that received IP DOX (Fig. 6A). In contrast, plasma drug levels were not significantly different between the two treatment groups on day 1 (Fig 6B). At 30 days post-treatment, DOX was detected only in local tissue of DoM-treated mice (211 ng/g) and undetectable elsewhere in either treatment groups. Additionally, across all times points, drug accumulation was generally lower in the major organs of DoM-treated mice compared to IP DOX-treated mice. Specifically, DOX levels in the heart were 47% lower on day 1 in DoM-treated mice compared to IP administration (Fig. 6C), 88% and 607% lower in the spleen (days 1 and 5, respectively) (Fig. 6D), 263% and 192% lower in the liver (days 1 and 5, respectively) (Fig. 6E), and 211% lower in the kidney (day 1) (Fig. 6F). No significant differences were detected in the lungs (days 1 and 5), the heart (day 5), and the kidney (day 5) (Fig. 6D,G,H). Of note, considerable splenic atrophy occurred in the IP DOX administered groups (0.028 ± 0.008 g with IP DOX vs 0.11 ± 0.015 g with DoM treatment) on day 5. This considerable decrease in organ weight coupled with high concentrations of drug within the spleen suggests acute toxicity from systemic (IP) treatment, but not from local treatment with the DoMs.

Fig. 6. Biodistribution of doxorubicin after mesh-delivery vs. systemic administration.

(A) Schematic of the studies. Concentration of DOX detected in (B) local tissue, (C) plasma, (D) heart, (E) spleen, (F) liver, (G) kidney, and (H) and lung at days 1, 5, and 30 after treatment with either single dose IP DOX or PCL-hybrid DOX (equivalent dose) film implantation. Representative image of the DoM on (I) day 1, (J) Day 5, (K) Day 30, (L) histogram representation of max detectable diffusion distance into adjacent tissue by day. *p<0.05, **p<0.01, unpaired T test.

To characterize the perfusion of DOX into tissue, we implanted DoMs in the dorsum of Nu/J mice and collected tissue on day 1, 5, and 30. Tissues were sectioned perpendicular to the mesh surface, stained with DAPI and WGA-488, and imaged via fluorescent microscopy (Fig. 6I-K, Fig. S6A-D). Peak diffusion distance was measured perpendicular to the mesh at 50 randomly distributed points. DOX was detectable up to 1.2, 2.6, and 3.3 mm into the tissue from the mesh surface on days 1, 5, and 30 respectively, with most of the DOX concentrated within 200-500 μm from the mesh (Fig. 6L).

DoMs do not induce pathophysiologic changes in cardiac function

While quantitative differences in DOX levels in vital organs is suggestive of improved safety, functional, clinically relevant outputs are necessary to demonstrate proof-of-concept for improved safety compared to systemic DOX administration. To determine if DoMs prevent cardiotoxic effects, we used echocardiography to assess physiologic differences in cardiac function between mesh-delivered DOX and IP DOX (500 μg or 20 mg/kg, respectively). After obtaining pre-treatment baseline echocardiographic measurements in non-tumor bearing mice, we repeated these measurements at days 5 and 20 post-treatment (Fig. 7A). Cardiac function significantly declined in IP DOX-treated mice as measured by cardiac output (Fig. 7B), ejection fraction (Fig. 7C), and fractional shortening (Fig. 7D) both at 5 and 20 days after treatment. Cardiac output (CO) decreased from baseline (23.0 ± 1.9 mL/min) by 37% by day 5 (15.1 ± 5.4 mL/min) and by 23% by day 20 (16.0 ± 2.7 mL/min). While cardiac output increased slightly from day 5 to day 20, the changes were not statistically significant (p = 0.79). Further, mice with the lowest cardiac output on day 5 were humanely euthanized prior to day 20, which skewed the mean results upwards. However, cardiac output declined in each individual mouse (Figure S7A). Ejection fraction (EF) also reduced from baseline (80.9 ± 4.9%) by 16% on day 5 (67.6 ± 13.1%) and by 23% on day 20 (62.6 ± 3.4%). Finally, fractional shortening (FS) declined in IP DOX-treated mice from baseline (49.1 ± 5.1%) by 23% on day 5 (37.6 ± 10.0%) and by 33% on day 20 (33.1 ± 2.3%). In contrast, cardiac function of the DoM-treated mice remained preserved, showing no statistical differences in CO, EF, and FS throughout the time course of observation compared to that of saline-treated controls. Additional measurements of ventricular dimension (systole and diastole), heart rate, end systolic and diastolic volumes, and left ventricular mass are found in Fig. S7B-G. In IP DOX-treated mice compared to DoM-treated mice, we observed a significant increase in systolic ventricular dimension (2.42 mm vs. 1.73 mm) (Fig. S7C), a significant decrease in heart rate (459 bpm vs. 622 bpm) (Fig. S7D), and a significant increase in end-systolic volume (0.021 mL vs. 0.009 mL) (Fig. S7F). There was no significant difference between the saline, IP DOX, and DoM-treated mice with respect to left ventricular dimension (Fig. S7B), end diastolic volume (Fig. S7E), and left ventricular mass (Fig. S7G). Together, these findings demonstrate that DOX delivery via DoM obviates a significant off-target and otherwise dose-limiting consequence of DOX treatment.

Fig. 7.

(A) Schematic of echocardiography protocol, (B) overall survival of mice after saline or a cardiotoxic dose of DOX delivered via IP injection or DoM, (C) cardiotoxicity evaluation as a measurement of cardiac output, (D) ejection fraction, and (E) fractional shortening, via echocardiography on days 5 and 20 after treatment of saline, a single dose IP DOX (20mg/kg), or PCL-hybrid DOX (500ug) film implantation. Solid bars represent median value of the data set. *p < 0.05, **p < 0.01, ***p < 0.001, unpaired T test.

Discussion

Local recurrence in soft tissue sarcoma occurs as the result of tumor cells remaining at the resection bed. While DOX is the first-line standard chemotherapy drug for the treatment of most advanced sarcomas, excluding those patients whose tumors are specifically resistant to DOX, its full anti-tumor potential is limited by poor drug delivery to the tumor site and dose-limiting side effects. To address these challenges, local delivery enables effective concentrations at the tumor site while minimizing accumulation in healthy tissue. Biomaterial drug delivery systems are widely investigated for localized delivery of chemotherapeutics due to their ability to encapsulate and release drugs at a controlled rate(36). To overcome the challenges associated with delivery of DOX after surgical resection, such as its hydrophilicity and short half-life, we hypothesized that an optimal drug delivery device must meet the specific design criteria discussed earlier.

Most local, extended-release chemotherapy formulations exist as rigid wafers, microparticles, or soft hydrogels(37). The biodegradable Gliadel^® wafer for the treatment of glioblastoma is the prime example (Eisai Inc.), and its use increases patient survival by 2.3 months(38). However, this rigid polymeric wafer releases over 50% of its drug cargo in 4 hours, over 70% in 24 hours, and the remaining drug just five days post-implantation(39). The burst release profile of Gliadel is not optimal for treatment of sarcomas, as only 11% of the tumor cells are in mitosis at any given time, and it causes acute toxicity in the local area(40). Furthermore, this brittle wafer is not amenable to suturing or stapling at the surgical site. Formulation plays a critical role controlling the drug release kinetics, and compressing polymer discs does not allow for augmentation, necessitating a method of preparation that is easily optimizable.

Given doxorubicin’s hydrophilicity, hydrogels represent a convenient means to package and subsequently deliver DOX, but these systems also suffer from burst release(41). Despite this drawback, several groups report hydrogels delivering DOX for the treatment of breast and liver cancer(42-44). Hydrogels are easily administered to a confined space such as the breast; however, their placement and attachment into an open and larger cavity, as in many cases of retroperitoneal soft tissue sarcoma, is challenging.

Textile meshes, commonly used in hernia and reconstruction surgery, are manufactured using easily scalable and optimizable technologies, such as electrospinning. However, DOX exhibits limited solubility in organic solvents necessary for electrospinning polymeric meshes. We utilize the free base, hydrophobic form of DOX to allow direct encapsulation of DOX in electrospun nanofibers or a polymer casting between the fibers. To construct the DOX-loaded polymeric meshes (DoMs), we selected PGA and PCL as the base polymer materials as both are GRAS (Generally Recognized As Safe) polymers. Additionally, we used the hydrophobic polymer PGC-C18 as a coating on the meshes to decelerate the drug’s release. PGC-C18 is biodegradable and successfully passed in vitro and in vivo biocompatibility testing according to ISO-10993 and FDA G95-1 guidelines with no measurable toxicity(45). Thus, our meshes are surgically implantable, fabricated via scalable manufacturing methods, and composed of safe, non-toxic, and biodegradable materials, achieving design requirements 1-3.

Varying key aspects of mesh architecture such as external coating, buttress material, and DOX loading strategy allows for tuning of the release rate and control of burst release. We explored loading DOX in the exterior coating and within the fibers to diminish burst release, and we employed a hybrid combination to achieve a small initial release followed by sustained release overtime. After resection, when microscopic tumor burden may be relatively high, DoMs eliminate remaining tumor cells with a sufficient bolus of drug and provide a steady concentration of DOX thereafter. The PCL-based meshes display prolonged DOX release due to greater mesh integrity, as the PGA-based meshes dissolve in vitro. Only the PCL-2-C/F film maintains its cytotoxic effect for 6-8 weeks in vitro due to the optimized release profile. These results demonstrate that DoMs release DOX for at least 4 weeks with a small burst release and retain DOX efficacy after encapsulation, accomplishing design requirements 4 and 5.

In vivo, the DoMs significantly increase time to recurrence and overall survival in two sarcoma resection models when compared to surgery alone, unloaded film, one-time bolus dosing of IP DOX, and weekly IP DOX treatments. 97% of mice in the control groups developed a local recurrence by 100 days. Between the LP6 and CS1 experiments, local recurrence occurred in only one mouse in the LP6 model after implantation of the DoM. Thus, our meshes prevent local recurrence in LP6 and CS1 models of sarcoma and increase overall survival in mice, fulfilling design requirement 6. Biodistribution data reveals that DoMs deliver significantly more DOX to the local tissue compared to IP administration. The DOX concentration in major organs is also lower after implantation of the mesh compared to the IP systemic dose, even though the mesh contains equal loading of DOX. This is particularly notable in cardiac tissue. On day 1, the ratio of total detected DOX in the distant organs compared to local tissue is 30.7 in mice receiving IP DOX compared to 0.57 in mice receiving DoMs. On day 5 these values are 21.7 and 1.26 for IP DOX and DoM groups respectively. This demonstrates that DoMs provide a high level of DOX to local tissue, but prevent accumulation of DOX in distant tissue relative to systemic administration. This represents a highly significant increase in the efficiency of DOX accumulation in the local tissue while avoiding high levels in distant healthy tissues.

Cardiomyopathy is the major toxic effect that limits DOX dosage for all cancer treatments, including sarcomas. Strategies for controlling cardiotoxicity include protocols for slow, continuous infusion to avoid bolus dosing(15,46) or reducing oxidative stress(47). There is a steep dose response curve regarding efficacy for DOX and soft tissue sarcoma, however, DOX-associated cardiomyopathy is also dose-dependent(48). Despite preventative limitations on dosing (<450 mg/m²), both acute (within 3 days) and chronic cardiotoxicity (within 30 days, up to 10 years) occur in patients receiving DOX (11% and 1.7% incidence, respectively)(49). Dexrazoxane is the only FDA-approved cardioprotective agent for use with DOX but can lead to severe myelosuppression so its use is limited(50). In our study, mice treated with IP DOX show echocardiographic evidence of cardiomyopathic toxicity at 5 and 20 days through decreased ejection fraction, cardiac output, and fractional shortening relative to untreated saline control. In contrast, mice treated with the same dose of DOX via DoM administration exhibit preserved cardiac function. These results document that DoMs eliminate cardiomyopathic toxicity associated with systemic DOX treatment, achieving our final design requirement. Improved efficacy without cardiotoxicity represents a significant improvement to available treatment regimens.

These studies demonstrate proof-of-concept that DoMs fulfill an unmet need in surgical oncology: reducing locoregional recurrence in soft tissue sarcoma. We accomplished this by fabricating, characterizing, and optimizing a combined surgical drug-device, and we demonstrated enhanced safety and efficacy in vivo. Local drug delivery via DoM improves survival and time to recurrence in two murine models of human sarcoma - an aggressive chondrosarcoma cell line and a patient-derived xenograft liposarcoma. DoMs afford sustained drug release kinetics with improved therapeutic dosing while preventing cardiotoxicity observed in mice receiving systemic doxorubicin. The study’s limitations include the use of murine hosts, human cell lines, and patient-derived xenografts, which do not perfectly recapitulate human disease. Further, sarcoma exhibits a diverse array of phenotypes, many of which cannot be captured by cell lines or xenografts, and few established models of recurrence in soft tissue sarcoma exist. In conclusion, the novel DOX-eluting mesh provides high local concentrations of drug for a sustained duration, improves survival in two murine sarcoma models, and avoids off-target side effects, specifically cardiotoxicity. The ability to deliver chemotherapy gradually using a biodegradable, scalable platform holds substantial clinical promise for sarcomas. Intended future work includes testing DoMs against other DOX-responsive cancers, evaluating the scalability and safety of meshes in larger animal models (e.g., mini pigs), utilizing meshes in combination with other therapies, and translating DoMs into the clinic for use in humans.