Improved I.P. Drug Delivery with Bioadhesive Nanoparticles

Improved i.p. drug delivery with bioadhesive nanoparticles

Yang Denga, Fan Yanga, Emiliano Coccob, Eric Songa, Junwei Zhanga,c, Jiajia Cuia, Muneeb Mohideena, Stefania Belloneb, Alessandro D. Santinb, and W. Mark Saltzmana,c,1

aDepartment of Biomedical Engineering, Yale University, New Haven, CT 06511; bDepartment of Obstetrics, Gynecology & Reproductive Sciences, School of Medicine, Yale University, New Haven, CT 06511; and cDepartment of Chemical & Environmental Engineering, Yale University, New Haven, CT 06511

Edited by Joseph M. DeSimone, University of North Carolina at Chapel Hill and Carbon, Chapel Hill, NC, and approved August 19, 2016 (received for review November 22, 2015) The i.p. administration of chemotherapy in ovarian and uterine (∼10–100 μm) and therefore, should distribute more evenly serous carcinoma patients by biodegradable nanoparticles may throughout the peritoneum. Furthermore, nanoparticles are small represent a highly effective way to suppress peritoneal carcinoma- enough to be internalized by tumor cells (17, 18), allowing them to tosis. However, the efficacy of nanoparticles loaded with chemo- release the encapsulated drugs near the biological target, which is therapeutic agents is currently hampered by their fast clearance by often in the cell nucleus. Although many studies have shown that lymphatic drainage. Here, we show that a unique formulation of nanoparticles are promising vehicles for the delivery of chemother- bioadhesive nanoparticles (BNPs) can interact with mesothelial apeutic agents in preclinical in vivo models, the application of cells in the abdominal cavity and significantly extend the retention nanoparticles for i.p. delivery remains hampered by their fast of the nanoparticles in the peritoneal space. BNPs loaded with a clearance from the peritoneal cavity, mainly as a result of lymphatic potent chemotherapeutic agent [epothilone B (EB)] showed signif- drainage (19). icantly lower systemic toxicity and higher therapeutic efficacy In a recent study (20), we described a novel form of bio- against i.p. chemotherapy-resistant uterine serous carcinoma- adhesive nanoparticles (BNPs) and showed that their adhesive- derived xenografts compared with free EB and non-BNPs loaded ness results in prolonged retention on the skin after topical with EB. delivery. These new materials are based on polylactic acid block– hyperbranched polyglycerol (PLA-HPG) copolymers, which can drug delivery | nanoparticles | ovarian cancer | intraperitoneal | be formed into “stealthy,” nonadhesive nanoparticles (NNPs) chemotherapy that circulate for long times after i.v. injection (21). The BNPs were produced by oxidation of the NNPs, which results in con- igh-grade ovarian and uterine serous carcinoma (USC) are version of vicinal diols on the surface of NNPs into aldehydes on Hbiologically aggressive tumors, which commonly spread into the BNPs (Fig. 1). Aldehydes spontaneously react with proteins the peritoneal cavity, disseminating along the abdominal and to form a variety of bonds, such as Schiff-base bonds; therefore, pelvic peritoneum and resulting in peritoneal metastases. Al- the presence of surface aldehydes on BNPs leads to particle though these tumors are often responsive to initial cytoreductive adhesion to protein-rich materials. For example, we previously surgery and platinum–paclitaxel chemotherapy, most patients showed the rapid and robust adhesion of the BNPs to poly-L- – develop recurrence and eventually die from progression of che- lysine coated surfaces and pig or mouse skin (20). In this work, motherapy-resistant disease (1). we hypothesize that BNPs will interact and adhere strongly with In cases of advanced ovarian cancer, the infusion of chemo- mesothelial cells layering the abdominal cavity after i.p. delivery therapeutic agents directly into the peritoneum (i.e., i.p. chemotherapy) improves overall and disease-free survival compared with Significance standard i.v. chemotherapy (2). However, despite level 1 evidence from meta-analyses of three large, prospectively randomized, Resistance to platinum-based chemotherapies and paclitaxel is controlled trials, this route of administration is still not widely used common in recurrence of both high-grade ovarian and endo- in the clinic because of its more severe side effects compared with metrial cancers. Paclitaxel resistance has been correlated with i.v. chemotherapy and the need for frequent dosing schedules (2, overexpression of class III β-tubulin, the preferential target of 3). To enhance the retention of chemotherapeutic drugs in the the epothilones, microtubule-stabilizing agents. Epothilone B peritoneal cavity and improve their bioavailability, controlled re- (EB) is manifold more effective than paclitaxel, but clinical use lease drug carriers, such as microparticles and hydrogels, have is limited by side effects. To reduce side effects, we encapsu- recently been tested in preclinical animal models (4–8). Although lated EB into bioadhesive nanoparticles (BNPs), reasoning that these carriers can lead to improvement over conventional delivery bioadhesive nanoparticles loaded with epothilone B (EB/BNPs) methods, both strategies only use the microparticles or hydrogel as would interact with abdominal tissues and gradually release EB reservoirs for drugs. To achieve expected therapeutic efficacy, the in proximity of peritoneal cancer implants, thus maintaining EB concentration of the free drugs in the peritoneal cavity has to be concentration at the site of action and limiting systemic ex- maintained in the effective range. Sustained drug concentrations posure and toxicity. Our experiments show the higher thera- require a balance between the drug release from carriers and clear- peutic activity and limited toxicity of EB/BNPs compared with ance from peritoneal cavity by metabolism. For a specific drug and nonadhesive nanoparticles loaded with EB or carrier-free EB. SCIENCES

drug carrier combination, release that is too slow might also com- APPLIED BIOLOGICAL Author contributions: Y.D., F.Y., E.C., E.S., J.Z., J.C., M.M., A.D.S., and W.M.S. designed promise the therapeutic efficacy as observed when limited disso- research; Y.D., F.Y., E.C., E.S., J.Z., J.C., M.M., S.B., A.D.S., and W.M.S. performed research; lution of paclitaxel from a microparticle/hydrogel system impaired Y.D., F.Y., E.C., E.S., J.Z., J.C., M.M., S.B., A.D.S., and W.M.S. analyzed data; and Y.D., E.C., therapeutic outcomes (5). In addition, side effects of some of these A.D.S., and W.M.S. wrote the paper. carriers do exist (i.e., microparticles accumulate in the lower ab- The authors declare no conflict of interest. domen and cause peritoneal adhesion) (9–11). This article is a PNAS Direct Submission. Degradable polymer nanoparticles have several potential advan- 1To whom correspondence should be addressed. Email: [email protected].

– ENGINEERING tages over microparticles and hydrogels in cancer therapy (12 16). This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Nanoparticles (∼100 nm) are small compared with microparticles 1073/pnas.1523141113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1523141113 PNAS | October 11, 2016 | vol. 113 | no. 41 | 11453–11458 Downloaded by guest on September 24, 2021 successfully used in prior work to generate consistent i.p. tumors in mice (28). Results In a previous work, we showed that NNPs had an extended blood circulation because of their resistance to nonspecific interaction to proteins (21). In another recent study, we found that BNPs had an enhanced interaction with protein-rich surfaces caused by their aldehyde groups (20). These results suggest that this class of nanoparticles can be converted from a nonadhesive to a strongly adhesive state and then back again by regulation of the density of aldehydes on the nanoparticle surface. To illustrate this flexi- bility, nanoparticles were converted by a cycle of oxidation and reduction between the hydroxyl-rich NNP state, the aldehyde-rich BNP state, and a reduced nonadhesive nanoparticle (NNP-R) state. We reduced the aldehydes to alcohols by treatment with a common reducing agent (29), NaBH4 (Fig. S1A). The concentration of aldehyde groups on BNPs was monitored as a function of duration of NaBH4 treatment; most aldehyde groups were converted after 2 min of treatment (Fig. 2D). It is important to note that NaBH4 treatment cannot reduce the aldehydes to the original vicinal diols, because one carbon is truncated by the initial conversion of vicinal diols to aldehydes (Fig. S1B). BNP, NNP, and NNP-R were identical in shape by transmission EM (TEM) (Fig. 2 A–C) and hydrodynamic size measured by dy- namic light scattering (DLS) (Table 1), suggesting that oxidation and reduction cycles had no detrimental effect to the nanoparticles. To quantitate bioadhesion, we tested nanoparticles at various states of aldehyde activation by examining attachment to poly(L-lysine)–coated plates and USC cells (Fig. 2 E and F). To facilitate quantification of their bioadhesive properties, all nanoparticles were loaded with 0.2% 1,1′-dioctadecyl- 3,3,3′,3′-tetramethylindodicarbocyanine, 4-chlorobenzenesulfo- nate salt (DiD) dye. We first evaluated the bioadhesive property of the three nanoparticles on poly(L-lysine)–coated surfaces. After incubation and extensive washing, the BNPs showed much higher retention on the poly(L-lysine)–coated plates (Fig. 2E). The BNPs also showed much higher attachment to USC cells (Fig. 2F). The retention of NNPs or NNP-Rs on both poly(L- lysine) surfaces and USC cell monolayers was negligible (Fig. 2 E Fig. 1. Schematic showing the conversion of BNPs from NNPs and the fate and F). We further tested the nanoparticles for blood circulation of NNPs and BNPs after i.p. delivery. BNPs can be converted from NNPs by times after i.v. injection in mice. The percentage dose of BNPs NaIO4 treatment. After i.p. delivery, (Left) NNPs are cleared by lymphatic dropped to 1.5% after 2 h, whereas significant numbers of NNPs drainage, but (Right) BNPs are retained in the peritoneal cavity because of (21.3%) were still circulating after 24 h (Fig. 2G). NNP-Rs cir- their bioadhesive property. culated with similar duration as the NNPs, reflecting the chemical similarity of their surfaces. (Fig. 1). We also hypothesize that the interaction of the BNPs We hypothesize that BNPs will adhere avidly to protein-rich with abdominal tissue will extend the retention of the BNPs after tissue surfaces and remain adherent for long periods. To test this i.p. injection, therefore significantly improving the bioavailability hypothesis, we first characterized the adhesive potential of BNPs of their payloads and the efficacy of i.p. chemotherapy. We and NNPs on human umbilical veins. Both DiD/NNPs and DiD/ tested the effectiveness of this approach for i.p. delivery of BNPs were applied individually to the luminal surface of human patupilone [epothilone B (EB)], a microtubule-stabilizing agent umbilical veins. After 2 h of incubation within the lumen fol- lowed by extensive washing by vessel perfusion with 30 mL PBS, that targets class III β-tubulin. Importantly, EB has been shown the BNPs showed much higher retention on the vessel endo- to be 3- to 20-fold more effective than Paclitaxel (PTX) in vitro thelium than NNPs (Fig. 3 A and B). Most significantly for this against multiple PTX-sensitive and -resistant human tumor cell lines – study, we evaluated the retention of nanoparticles within the (22 25). Unfortunately, the clinical use of EB in patients with peritoneum after i.p. administration in mice. In this case, BNPs recurrent disease is limited because of high toxicity, including and NNPs were loaded with 0.5% IR-780 iodide (IR-780) dye to severe diarrhea, vomiting, increased risk of bowel obstruction, facilitate imaging. At this loading, only a small fraction of the IR- and fatigue (26, 27). Here, we use our BNPs loaded with EB to 780 dye is released from the nanoparticles, and it is released test whether strongly adhesive nanoparticles will improve the slowly over 10 d of incubation in buffered saline supplemented efficacy of i.p. chemotherapy to PTX-resistant peritoneal me- with physiological levels of protein (Fig. S2A); therefore, the dye tastasis. In particular, we developed i.p. xenograft models de- molecule serves as a reliable marker for the presence of nano- rived from the primary USC cell line USC-ARK-2. This cell line particles. As we have shown with other NNP/BNP combinations, was selected (i) because of its high expression of P-glycoprotein the conversion of IR-780/NNPs to IR-780/BNPs (by treatment β and tubulin- -III, characteristics that have been recently shown with NaIO4) had no apparent effect on nanoparticle size and mor- by our group to confer PTX resistance while showing extreme phology as observed by TEM (Fig. S3) or hydrodynamic size as sensitivity to EB (25), and (ii) because this cell line has been measured by DLS (Table 1). After i.p. injection of NNPs, most of

11454 | www.pnas.org/cgi/doi/10.1073/pnas.1523141113 Deng et al. Downloaded by guest on September 24, 2021 Fig. 2. TEM images of (A) DiD/NNPs, (B) DiD/BNPs, and (C) DiD/NNP-Rs (DiD/NNPs reduced from DiD/BNPs by NaBH4 treatment). (Scale bars: 200 nm.) (D) The concentration of aldehydes on BNPs as a function of incubation time with NaBH4.(E) Retention of BNPs on (poly-L-lysine)–coated plates compared with NNPs and NNP-Rs. *P < 0.05 compared to both other groups. (F) Retention of BNPs on USC cell monolayers compared with NNPs and NNP-Rs. *P < 0.05 compared to both other groups. (G) The percentage of dose of DiD/NNPs, DiD/BNPs, and DiD/NNP-Rs in blood as a function of time after i.v. injection in mice.

the fluorescent signal, which was initially strong (5 min), was EB/BNPs, EB/NNPs, and free EB were separately added to USC lost by the end of the first day (Fig. 3C). In contrast, after i.p. cell cultures, which were tested for viability after 3 d of exposure. injection of BNPs, a strong fluorescent signal was observed to All EB treatments (EB, EB/NNPs, and EB/BNPs) yielded simi- last up to 5 d (Fig. 3C); at the end of the 10th day, BNPs were lar dose–response curves, with the free EB showing the highest still detectable in the area of the initial injection (Fig. S2B). toxicity (i.e., lowest IC50) (Fig. 4D). We believe that this differ- Remarkably, accumulation of BNPs in the lower abdomen, ence between the free EB and nanoparticle formulations of EB which is commonly seen after microparticle i.p. delivery (30), is caused by the slow release of EB from NNPs and BNPs, was not observed with administration of BNPs; the major- resulting in a slightly lower concentration of EB in the culture ity of the BNPs adhered to the parietal peritoneum of the medium exposed to nanoparticle formulations compared with mice (Fig. S4). free EB. To confirm that the cytotoxicity was caused by the EB To determine the efficacy of nanoparticles as vehicles for i.p. and not the polymers within the nanoparticles, we also incubated delivery, we loaded NNPs and BNPs with EB. Nonadhesive USC cells with unloaded (blank) BNPs and NNPs. Unloaded nanoparticles loaded with epothilone B (EB/NNPs) and bio- nanoparticles did not show any toxicity, even when added to the adhesive nanoparticles loaded with epothilone B (EB/BNPs) culture medium at high concentrations (1 mg/mL) (Fig. 4E). The were of similar hydrodynamic size, ∼130 nm in diameter (Table tolerable concentration of blank nanoparticles is much higher 1). EB/BNPs and EB/NNPs were spherical, with no differences than the nanoparticle concentration of EB/NNPs or EB/BNPs in morphology as observed by TEM (Fig. 4 A and B). Both NNPs needed to deliver an effective dose: for example, 10 nM EB is and BNPs had a loading of EB of 2% by weight. When EB/NNPs contained within 0.25 μg/mL EB/NNPs or EB/BNPs. To further and EB/BNPs particles were incubated in PBS at 37 °C, the show the safety of the nanoparticle treatments, we examined the majority (about 80%) of the EB was released during the initial cytotoxicity of blank BNPs and NNPs on human umbilical vein 8 h of incubation (Fig. 4C). To test for in vitro cytotoxicity, endothelial cells and HeLa cells. Neither BNPs nor NNPs were toxic in these cells up to 1 mg/mL (Fig. S5A). Furthermore, we incubated blank BNPs or NNPs at 1 mg/mL on a variety of im- Table 1. Nanoparticle size measurements mortalized cell lines and observed no cytotoxicity at high con- SCIENCES Nanoparticles Diameter (nm) Polydispersity index centration (Fig. S5B). To measure retention of EB within the peritoneal tissue, we i.p. administered free EB, EB/BNPs, and EB/NNPs 127 0.225 EB/NNPs in nude mice. No EB was detected in mice receiving EB/BNPs 127 0.233 the free EB at 6 h after a single dose (Fig. 4F). In contrast, DiD/NNPs 121 0.215 detectable levels of EB were found at 6 h with both EB/NNPs DiD/BNPs 117 0.232 and EB/BNPs injections: the EB/BNPs-treated mice had sub- DiD/NNP-Rs 97 0.213 stantially higher concentrations of EB than the EB/NNPs-treated IR-780/NNPs 131 0.331 mice (21% vs. 5%, P < 0.05) (Fig. 4F). One day after i.p. ad- ENGINEERING APPLIED BIOLOGICAL IR-780/BNPs 128 0.297 ministration, EB was still significantly higher after EB/BNPs

Deng et al. PNAS | October 11, 2016 | vol. 113 | no. 41 | 11455 Downloaded by guest on September 24, 2021 (P = 0.03) or EB, with 60% of the animals surviving longer than 110 d (Fig. 6C). As expected, the survival rate for mice treated with blank BNPs was indistinguishable from that of mice treated with PBS (P = 0.83) (Fig. 6C). No significant difference was observed between mice treated with EB/NNPs and mice treated with free EB (P = 0.32), both with ∼10% of the animals surviving to 110 d. Discussion In this study, we have synthesized BNPs that interact with peritoneal tissues, dramatically enhancing their duration of retention in the abdominal cavity after i.p. administration. Previous studies have shown that the diameter of the lymphatic ducts in the peritoneal cavity is about 1 μm. Microparticles, which typically have diameters larger than 4 μm, can escape the lymphatic drainage, resulting in long-lasting retention after i.p. administration (4, 31). However, microparticles tend to accumulate in the lower abdomen because of gravitational forces (30), likely leading to regions of high and low drug concentration. This heterogeneous distribution may also cause potential serious side effects, including inflammation and peritoneal adhesion (9–11), limiting their clinical application. Although multiple studies have Fig. 3. Imaging of DiD/BNPs and DiD/NNPs ex vivo and in vivo. (A) DiD/BNPs shown the advantages of nanoparticles in the delivery of drugs and DiD/NNPs were incubated at 1 mg/mL in Ringer’s buffer within the lu- and biologic agents, the use of nanoparticles in the abdominal men of umbilical veins for 2 h at 37 °C. The red fluorescence signal represents cavity is limited because by their rapid clearance caused by their nanoparticles retained on the luminal surface of the vein after extensive small size (∼100 nm) (19). Here, we show that the addition of a washing. (B) Quantification of fluorescence retained on vein. Data are dense coating of aldehyde groups on the particle surface confers ± < shown as means SD. P 0.0001 (Student t test). (C ) The retention of bioadhesive properties to the nanoparticles (converting them the IR-780/NNPs and IR-780/BNPs monitored with live imaging after i.p. administration. into BNPs). This adhesive property promotes nanoparticle interaction with tissues, therefore extending their retention in the abdomen after i.p. injection (Fig. 3). Based on an in vitro model injection compared with EB/NNPs injection (P < 0.05). No EB study (Fig. 5), we believe that this prolonged retention of BNPs was detectable after 3 d for all formulations. leads to improved treatment of disseminated tumors in the OurhypothesisisthatEB/BNPscanprotecttheperitoneum abdominal cavity. Consistent with this hypothesis, free EB and against the attachment of floating cancer cells in the peritoneal fluid; this protection is provided by BNP retention on protein-rich peritoneal surfaces and their ability to deliver EB locally to treat adherent cells (Fig. 1). To simulate the microenvironment in which we expect the nanoparticles to reside after i.p. injection, we evaluated the in vitro efficacy of surface-immobilized EB/BNPs to suppress the growth of USC cells. Here, we used microscope slides coated with poly(L-lysine) and incubated them with free EB, unloaded nanoparticles, or EB-loaded nanoparticles (Fig. 5A). We observed significant suppression of tumor cell growth only in slide regions pretreated with EB/BNPs (Fig. 5 B and C). No suppression of tumor growth was observed on surfaces treated with either blank BNPs or EB/NNPs. Although this is an in vitro model system and lacks many of the features of the i.p. environment, these results suggest that BNPs are retained on the lysine-coated surface of the slides because of their bioadhesive properties and able to deliver EB locally to adjacent cells more effectively than any of the other nanoparticle preparations. We evaluated the safety and efficacy of EB treatments in nude mice harboring class III β-tubulin overexpressing USC xenografts in the peritoneal cavity. One week after tumor inoculation, EB/ BNPs, EB/NNPs, and free EB were administrated weekly at an EB dose of 2.5 (Fig. 6 A and B) or 0.5 mg/kg (Fig. 6 C and D). Our intent was to treat animals in both experiments for 5 wk, but animals receiving the higher dose of free EB (2.5 mg/kg) experienced weight loss (Fig. 6B) and early death caused by EB toxicity (Fig. 6A); therefore, all animals in the high-dose (2.5 mg/kg) experiment were treated for only 3 wk. At both doses, mice treated with EB/BNPs survived, on average, significantly longer Fig. 4. Characterization of EB/BNPs. TEM images of (A) EB/BNPs and (B)EB/ than control animals treated with PBS (P = 0.0006, 2.5 mg/kg; NNPs. (Scale bars: 200 nm.) (C) EB is released from EB/BNPs and EB/NNPs over ± = P = 0.0005, 0.5 mg/kg), but in mice treated with free EB, only a period of 24 h of incubation in PBS. Data are shown as means SD (n 4). P = (D) Viability of USC cells after incubation with free EB, EB/NNPs, and EB/BNPs mice treated with the lower dosage showed significance ( 0.17, ± = P = A C for 3 d. Data are shown as means SD (n 8). (E) Viability of USC cells after 2.5 mg/kg; 0.01, 0.5 mg/kg) (Fig. 6 and ). Especially incubation with blank BNPs and NNPs for 3 d. Data are shown as means ± SD of note, mice treated with EB/BNPs (0.5 mg/kg) survived, on (n = 8). (F) EB retention in the peritoneal cavity after i.p. administration of average, significantly longer than mice treated with EB/NNPs free EB, EB/NNPs, and EB/BNPs. Data are shown as means ± SD (n = 3).

11456 | www.pnas.org/cgi/doi/10.1073/pnas.1523141113 Deng et al. Downloaded by guest on September 24, 2021 suitable for i.p. therapy. We showed the effectiveness of these EB formulations by measuring the survival of mice with PTX-resistant USC xenografts. To further limit EB toxicity in animals, we used a fivefold lower dose of EB compared with previous studies in nude mice with human multiple myeloma xenografts (37). Even at this lower dose of EB, however, significant toxicity (i.e., weight loss, diarrhea, and bowel dilation) was observed in mice treated with free EB. Remarkably, very little toxicity was observed in the mice treated with EB/BNPs (Fig. 6B). We attribute this difference in toxicity to two factors associated with EB/BNPs: the gradual release of EB from the BNPs (Fig. 4C), which likely reduces peak drug levels, and the strong adhesion of BNPs to the peritoneal tissue (Fig. 3C), which likely reduces EB doses to nontarget tissues and vital organs by fast lymphatic drainage, although some local toxicity to adjacent normal tissue is likely unavoidable (although not observed in our studies). We showed that EB was quickly cleared from the abdominal cavity, likely by diffusion through the peritoneal membrane into capillaries and transport through the hepatic portal system (38), which would be expected to lead to side effects and significantly lower bioavailability (39). The main side effect caused by EB in our Fig. 5. Suppression of USC cell growth on EB/BNPs-treated surfaces. study was gastrointestinal toxicity, which is in agreement with the (A) Schematic showing experimental design. A glass slide was divided into known toxicity profile of EB (26, 40, 41). five blocks, and each block was incubated individually with EB/BNPs, EB/ Importantly, in the EB/BNPs-treated mice, we showed signifi- NNPs, free EB, BNPs, or PBS for 30 min. After extensive wash, the slide was cantly improved survival compared with in the controls (P < 0.05), incubated in RPMI medium with USC cells with density at 2.0 × 105/mL for 24 h. After wash, the USC cells on slides were stained with Hoechst (for nuclei; with 60% of the animals alive at the end of the experiment (110 d). blue) and live/dead stain (green indicates live cells, and red indicates dead We attribute this enhanced effectiveness to EB/BNP bioadhesion cells) and imaged with a fluorescence microscope. (B) Fluorescence images and slow release. As evidence for the importance of bioadhesion, showing the USC cell growth on poly-(L-lysine)-coated slides pretreated with EB/NNPs were not as effective as EB/BNPs. different EB formulations. (Scale bars: 1 mm.) (C) The surface density of the Recent studies have shown strong activity of small molecules that cells was quantified with ImageJ and normalized to the PBS control. Data are selectively target the Her2/PI3K/Akt/mTor pathway against USCs. shown as means ± SD (n = 3). *P < 0.05. However, treatments with a single agent were only transiently effective, and tumors rapidly acquired resistance in vivo (36, 42). Importantly, recent preclinical data suggest that dual targeting of EB/NNPs were rapidly cleared from the peritoneal cavity, whereas HER2/PIK3CA with a Pan-HER inhibitor (Neratinib) and a EB was retained much longer when administered in EB/BNPs PIK3CA inhibitor (Taselisib) may be synergistic and able to achieve (Fig. 4F). Our measurement of release from BNPs in vitro (∼90% durable regression of USC xenografts in mice (43). However, this released in 24 h) (Fig. 4C) is consistent with the drug retention observed in vivo (∼5% retained at 24 h) (Fig. 4F), despite the fact strategy is only applicable for the treatment of patients whose USC tumors harbor the amplification of the c-erbB2 gene and the con- that the in vitro release was measured in an artificial system in PI3KCA which the nanoparticles were suspended in buffered saline, which comitant amplification or oncogenic mutations in the gene. is not identical to the conditions experienced by BNPs in the Therefore, alternate strategies are likely needed. In this study, we peritoneal space. We believe that slow EB release over 24 h leads propose the use of nanoparticles loaded with EB for the treatment to the reduced EB toxicity and enhanced EB effectiveness (Fig. of USC after local/regional (i.p.) administration. This strategy takes 6A). The prolonged retention time that we observed for BNPs advantage of the chemical properties of the nanoparticles and the (some particles are retained for 5–10 d) (Fig. 3C and Fig. S2B) may not be needed in this particular case, but this long retention may prove to be useful in other applications: for example, for BNPs loaded with other drugs that are released over longer periods. When not loaded with drug agents, the BNPs are not toxic. In cell cultures, BNPs did not show any cytotoxicity, even at high concentration (i.e., up to 1 mg/mL), and mice treated with BNPs (i.p. injection of 5 mg BNPs once a week for 5 wk) had no evidence of weight loss or behavioral changes. In addition, the repeated i.p. injection of BNPs did not seem to lead to any nonspecific responses that affected tumor growth or animal health (Fig. 6). We attribute the low toxicity of BNPs to several factors. (i) Although free low- molecular weight aldehydes can be toxic (32), the surface aldehyde SCIENCES groups are covalently attached to BNPs, limiting their dispersion. (ii) High tolerance to aldehydes is expected, because they are widely present in foods, fragrances, and metabolites and can be efficiently detoxified by the enzyme aldehyde dehydrogenase (33). Fig. 6. Therapeutic efficacy of EB/BNPs on mice bearing i.p. USC tumors. – In these studies, we selected EB as an agent for use in i.p. ad- (A)Kaplan Meier survival curves for EB treatments at 2.5 mg/kg. (B)Asa measure of toxicity, mice treated as in A were weighed twice a week. ministration. EB has remarkable efficacy against PTX-resistant – – (C)Kaplan Meier survival curves for EB treatments at 0.5 mg/kg. (D)Mice uterine serous and ovarian cancers (34 36), but prior clinical treated as in C were weighed twice a week. In A and C,thex axis indicates testing has revealed substantial toxicity when EB is administered thenumberofdaysfromthefirstdoseofdrug.InB and D, the average as a free drug (26). We hypothesized that encapsulation in weight is displayed as a function of days from the first drug dose. Statis- ENGINEERING APPLIED BIOLOGICAL nanoparticles would reduce the toxicity of EB and make it tical analysis was performed as described in SI Methods.

Deng et al. PNAS | October 11, 2016 | vol. 113 | no. 41 | 11457 Downloaded by guest on September 24, 2021 extreme sensitivity of USCs to the EB and may, therefore, be used emulsion–evaporation technique and characterized using TEM and DLS. for the treatment of the vast majority of USC patients, regardless of BNPs were formed using a previously described method (20) of using the genetic landscape of their tumors (44). NaIO4 (Fig. 1) to convert vicinal diols to aldehyde groups and reversing In summary, our results represent a demonstration that i.p. ad- back to NNP-Rs by adding NaBH4 (Fig. S1A). Retention of fluorescent ministration of BNPs encapsulating EB can enhance survival of BNPs was tested in vitro using a lysine-coated plate and wells containing USC cells by washing wells after particle incubation and reading the mice with i.p. tumors formed from USC xenografts. Importantly, measurements on a fluorescent plate reader. Ex vivo retention was the unique formulation of EB/BNPs exhibits higher therapeutic evaluated using a human umbilical cord and visualized with a fluorescent efficacy and lower toxicity than free EB and EB/NNPs. Because the microscope. In vivo retention, distribution, and efficacy of dye- and drug- bioadhesive property of BNPs is based on the general interaction loaded particles were tested in C57BL/6 and nude mice with USC cancers. between aldehydes on nanoparticles and proteins on tissue—which Characterization was completed with the use of an in vivo imaging sys- is illustrated in the variety of systems that we studied here (Figs. 2 tem (Xenogen), and efficacy was measured with strict survival guidelines. and 3A)—we expect this approach to be applicable to other strat- All animals were handled in accordance with the policies and guidelines egies for local delivery of active agents. of the Yale Institutional Animal Care and Use Committee. Detailed experimental procedures are provided in SI Methods. Methods

We used previously characterized BNPs to overcome barriers present ACKNOWLEDGMENTS. We thank Yukun Pan, Tian Xu, Yu Wu, Yao Lu, and in i.p. delivery of therapeutics. PLA-HPG was synthesized as previously Rong Fan of Yale University for access to instruments in their laboratories. reported (21). NNPs loaded with DiD, IR-780, and EB were formed using an This work was supported by NIH Grants CA149128 and CA154460.

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