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. 2021 Feb 16;118(7):e2020575118.
doi: 10.1073/pnas.2020575118.

Nonsurgical treatment of skin cancer with local delivery of bioadhesive nanoparticles

Jamie K Hu  1 , Hee-Won Suh  2 , Munibah Qureshi  1 , Julia M Lewis  1 , Sharon Yaqoob  1 , Zoe M Moscato  2 , Sofia Griff  2 , Alison K Lee  1 , Emily S Yin  1 , W Mark Saltzman  2 , Michael Girardi  3
Affiliations

Nonsurgical treatment of skin cancer with local delivery of bioadhesive nanoparticles

Jamie K Hu et al. Proc Natl Acad Sci U S A. .

Abstract

Keratinocyte-derived carcinomas, including squamous cell carcinoma (SCC), comprise the most common malignancies. Surgical excision is the therapeutic standard but is not always clinically feasible, and currently available alternatives are limited to superficial tumors. To address the need for a nonsurgical treatment for nodular skin cancers like SCC, we developed a bioadhesive nanoparticle (BNP) drug delivery system composed of biodegradable polymer, poly(lactic acid)-hyperbranched polyglycerol (PLA-HPG), encapsulating camptothecin (CPT). Nanoparticles (NPs) of PLA-HPG are nonadhesive NPs (NNPs), which are stealthy in their native state, but we have previously shown that conversion of the vicinal diols of HPG to aldehydes conferred NPs the ability to form strong covalent bonds with amine-rich surfaces. Herein, we show that these BNPs have significantly enhanced binding to SCC tumor cell surfaces and matrix proteins, thereby significantly enhancing the therapeutic efficacy of intratumoral drug delivery. Tumor injection of BNP-CPT resulted in tumor retention of CPT at ∼50% at 10 d postinjection, while CPT was undetectable in NNP-CPT or free (intralipid) CPT-injected tumors at that time. BNP-CPT also significantly reduced tumor burden, with a portion (∼20%) of BNP-CPT-treated established tumors showing histologic cure. Larger, more fully established PDV SCC tumors treated with a combination of BNP-CPT and immunostimulating CpG oligodeoxynucleotides exhibited enhanced survival relative to controls, revealing the potential for BNP delivery to be used along with local tumor immunotherapy. Taken together, these results indicate that percutaneous delivery of a chemotherapeutic agent via BNPs, with or without adjuvant immunostimulation, represents a viable, nonsurgical alternative for treating cutaneous malignancy.

Keywords: chemotherapy; drug delivery; immunotherapy; nanoparticle; squamous cell carcinoma.

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Conflict of interest statement

Competing interest statement: W.M.S. and M.G. have an ownership interest in and are paid consultants with Stradefy Biosciences, Inc.

Figures

Fig. 1.
Fig. 1.
Synthesis and bioadhesion of CPT-loaded PLA-HPG NNPs and BNPs. (A) NP synthesis schematic. (B) Transmission electron microscopy images of NNP-CPT and BNP-CPT particles. Following NNP oxidation with sodium periodate, BNPs retain their spherical morphology, indicating that conversion does not compromise the structure of the BNPs. (Scale bar: 100 nm.) (C) CPT-loaded NNPs and BNPs show similar temporal drug release kinetics in 1× PBS at 37 °C. Both NNP-CPT and BNP-CPT show the gradual release of drug over 24 h. (D) Quantification of bioadhesion of NNPs and BNPs in proteinaceous environments following rigorous washing. When quantifying bioadhesion, (E) BNP-CPT demonstrated superior adherence compared to NNP-CPT. Data normalized to unwashed controls. ****P < 0.0001, Student’s t test; n = 5.
Fig. 2.
Fig. 2.
BNPs readily associate with tumor cells. (A) Confocal microscopy of PDV SCC cells following 24-h incubation with DiD dye-loaded NNPs and BNPs demonstrate superior affinity of BNPs to tumor cells. (Scale bar: 20 μm.) (B) Assessment of cellular association over time via flow cytometric analysis of PDV cultured under serum-free conditions with Cy5-conjugated NPs (Left) and DiD-loaded NPs (Center) demonstrates significantly improved association of BNP in both cases, with their advantage especially notable at later time points (16 to 24 h). Baseline fluorescence (Right) of both dye-conjugated (Cy5) and -loaded (DiD) NNP and BNP before incubation is similar. A comparison of NP association with tumor cells following short (up to 5 h) incubation in serum-containing vs. serum-free conditions is shown in SI Appendix, Fig. S2. (C) Cellular association of Cy5-NPs (Left) and DiD-NPs (Right) with extended incubation in serum-containing media. Even with prolonged exposure, BNPs associate with tumor cells more strongly than NNPs. MFI, mean fluorescence intensity; n = 3 replicates. ***P < 0.001, Student’s t test.
Fig. 3.
Fig. 3.
BNPs facilitate both active internalization by tumor cells and binding to extracellular proteins to promote cytotoxicity. (A) Effects of temperature on NNP-Cy5 (Left) and BNP-Cy5 (Right) association with tumor cells. PDV cells cultured with either NNP-Cy5 or BNP-Cy5 at 4 °C in serum-containing media show only minimal cellular association, whereas 37 °C cultures show significant NP association over time, suggesting active cellular processes are required. MFI, mean fluorescence intensity; n = 3. (B) Interrogation of clathrin- or macropinocytosis-mediated mechanisms of cellular uptake. Cells were cultured in serum-containing media with NNP-Cy5 or BNP-Cy5 for 16 h and during the final 6 h of culture, vehicle (DMSO, 0.1%), CPZ (20 µM), or EIPA (50 µM) added. Results are expressed as the percent inhibition of NP uptake relative to vehicle control; n = 3. (C) Poly-l-lysine–coated plates were incubated with blank (unloaded) NP, free CPT, NNP-CPT, or BNP-CPT for 1 h. These solutions were then aspirated and PDVC57 seeded in fresh media and cultured for 48 h, when viability was assessed. (D) PDVC57 was incubated with free CPT, NNP-CPT, or BNP-CPT for 1 h. The CPT solutions were then aspirated and replaced with fresh media for 48 h, when viability was assessed. BNP-CPT increased tumor cell cytotoxicity compared to all other groups; n = 3. *P < 0.05, **P < 0.01, and ****P < 0.0001, Student’s t test.
Fig. 4.
Fig. 4.
BNPs exhibit greater association with tumor microenvironments in vivo to maintain local drug concentrations. Confocal microscopy of PDVC57 tumors and overlying skin 72 h after injection of NNP-Cy5 (A) or BNP-Cy5 (B). BNP-Cy5 exhibited greater intratumoral retention when compared to NNP-Cy5 (fluorescence integrated density/tissue area: NNP, 3,541.88; BNP, 10,402.26). (Scale bar: 200 μm.) (C) CPT recovered from tumors after treatment with intralipid-CPT (IL-CPT), NNP-CPT, or BNP-CPT. At 48 and 240 h following treatment, a greater percentage of the injected CPT was recovered from tumors treated with BNP-CPT (48 h, 72.4%; 240 h, 44.7%) when compared to free CPT (48 h, nd [not detected]; 240 h, nd) and NNP-CPT (48 h, 13.4%; 240 h, nd), indicating enhanced BNP-CPT association within SCC tumors. **P < 0.01, ****P < 0.0001, Student’s t test; n = 5.
Fig. 5.
Fig. 5.
Treatment of murine SCC with intratumoral BNP-CPTs is superior to NNP-CPT or intralipid-CPT. (A) Experiment timeline. Tumors injected with BNP-CPT showed significantly delayed tumor growth (B) and reduced tumor weight at harvest (C) (SI Appendix, Fig. S4) when compared to groups treated with either blank BNPs, IL-CPT, or NNP-CPT. (D) Combination therapy with BNP-CPT (12.5 mg/kg CPT) plus the TLR9 agonist, CpG (10 μg), significantly delayed tumor growth. *P < 0.05, **P < 0.01, Student’s t test (C), Mantel–Cox test (B and D; n = 10).
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