PARP-inhibitor nanoparticles and methods thereof

Abstract

This present disclosure is directed to poly (ADP-ribose) polymerase (PARP) inhibitor nanoparticle composition and methods of use and preparation thereof. PARP inhibitor dose reduction through combination treatment may be achieved using photodynamic therapy or photoimmunotherapy.

Description

PARP-INHIBITOR NANOPARTICLES AND METHODS THEREOF by Huang Chiao Huang Aaron Sorrin Kathryn McNaughtonCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Application No. 63 / 515,340, filed on July 24, 2023, the contents of which are hereby incorporated by reference in its entirety.

[0002] STATEMENT OF FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0003] This invention was made with government support under Grant No. R01CA260340 awarded by NIH, R01CA256710 awarded by NIH, and 2030253 awarded by NSF. The government has certain rights in the invention.

[0004] FIELD OF THE INVENTION

[0005] The field of the invention relates generally to PARP inhibitors and more particularly to the use of PARP inhibitors for oncologic applications.

[0006] BACKGROUND

[0007] The poly(ADP-ribose) (PAR) post-translational modification is a major biological regulator with broad roles in cell survival, gene expression, and energy metabolism. The transfer of PAR chains to target proteins is accomplished by PAR polymerases (PARPs), which use NAD+as a substrate and generate nicotinamide as a byproduct. PARP-1 binds to DNA single-strand breaks (SSBs), initiating PARylation of acceptor proteins including PARP-1, histones, and other DNA repair proteins. These appended PAR chains recruit additional DNA repair molecules for SSB rectification, such as X-ray repair crosscomplementing protein 1 (XRCC1). In recent years, there has been increasing clinical interest in PARP inhibitors (PARPi) for oncologic applications, particularly in patients with BRCA mutations where synthetic lethality can be achieved. Mechanistically, PARPi function through 1) directly competing with NAD+at the PARP catalytic site and 2) trapping PARP at the SSB site, forming toxic PARP -DNA complexes.

[0008] PARPi first entered the clinical sphere in 2014 with the United States Food and Drug Administration (FDA) and European Medicines Agency (EMA) approvals of olaparib forthe treatment of advanced ovarian cancer. Since then, three additional PARPi (rucaparib, niraparib, and talazoparib) have been FDA-approved for clinical use. These agents have been used for the treatment of numerous malignancies including ovarian, breast, pancreatic, prostate, fallopian, and primary peritoneal cancers. Talazoparib, the most recently FDA- approved PARPi, exhibits the greatest potency compared to olaparib, rucaparib, and niraparib, with lower half maximal inhibitory concentration (IC50) values for PARPs-1, -2, - 3, -4, and the strongest PARP trapping capabilities. While talazoparib is the most potent PARPi, it is also the most toxic to normal cells and the most poorly tolerated. As a result, maximum tolerated dose of talazoparib is at least 300-fold lower than that of other clinically used PARPi. There is a critical need to develop therapeutics to effectively deliver PARPi.

[0009] SUMMARY

[0010] One aspect of the invention pertains to a poly (ADP-ribose) polymerase (PARP) inhibitor nanoparticle composition, said composition comprising a PARP inhibitor (PARPi) and a polymer, wherein said composition is in the form of a nanoparticle particle.

[0011] Another aspect of the invention pertains to a method of preparing a PARP inhibitor nanoparticle composition, said method comprising: dissolving a PARP inhibitor and a polymer in a water-miscible solvent (e.g., acetone) to obtain a PARP inhibitor-polymer solution; and adding surfactant-containing water to obtain a mixture and sonicating said mixture; removing said solvent (e.g., evaporation) to obtain said PARP inhibitor -polymer nanoparticle.

[0012] A further aspect of the invention pertains to a method for the treating cancer, said method comprising administering a nanoparticle composition disclosed herein and optionally photosensitizer to a subject.

[0013] A further aspect of the invention pertains to a method for the treating cancer, said method comprising administering a nanoparticle composition disclosed herein and a photosensitizer disclosed herein, to a subject.

[0014] BRIEF DESCRIPTION OF THE FIGURES

[0015] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention.

[0016] FIGS. 1A-1Q. Schematic and characterization of talazoparib-loaded nanoparticles. NP-Tal is prepared by co-dissolution of polymers and talazoparib in acetone, which is added into surfactant-containing water. The mixture is sonicated and solvent is evaporated under constant spinning for 4-6 hours, then nanoparticles are concentrated using 30 kDa MWCO centrifugal filter FIG. 1 A. Representative TEM images are shown FIG. IB. Scale bar: 250 pm. During optimization procedures, initial talazoparib (mg) was varied (FIG. C-G) and initial PLGA-PEG-COOH (mg) was varied (FIG. H-L). (FIG. M-Q) Next PLGA-PEG-DBCO was mixed in with PLGA-PEG-COOH at varying amounts from 0-100% where total polymer mass remained fixed at 42.8 mg. The characterized parameters include loading capacity (%), encapsulation efficiency (%), size (nm), poly dispersity index, and zeta potential. * p < 0.05; **p < 0.01; *** p < 0.001; **** p < 0.0001.

[0017] FIG. 2A - 2K. Optimization and characterization of PIC-conjugated nanoparticles. Azide-functionalized photoimmunoconjugates were conjugated to DBCO-containing nanoparticles via copper-free click chemistry (FIG. 2A) and visualized by cryoEM (FIG. 2B). Scale bar: 500 pm. Volume ratio of PIC:NP was varied and changes in size (FIG. 2C), PIC conjugation efficiency (FIG. 2D), and number of PICs per nanoparticle (FIG. 2E) were characterized. The absorbance spectra are shown for BPD-containing formulations (FIG. 2F) and talazoparib-containing formulations (FIG. 2G) from 300-800 nm in DMSO. Next, comparison of 690 nm absorbance was performed in DMSO versus PBS (FIG. 2H). Photoactivity was evaluated, as described in Materials & Methods, for BPD, PIC, PIC-NP, and PIC -NP-Tal (FIG. 21). Singlet oxygen was next quantified using SOSG (Invitrogen) (FIG. 2 J). Uptake of 1 pM PIC versus 1 pM PIC-NP-Tal in EGFR-negative J774 cells compared to EGFR-positive OVCAR8 cells after 30 minute incubation (FIG. 2K). Data is normalized to J774 uptake of PIC. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

[0018] FIG. 3A - 3H. Optimization of 3D coculture model. OVCAR8-DsRed2 and NCI / ADR-RES-EGFP cells were plated at a 1 : 1 ratio to final seeding cell densities of 1,000- 5,000. Fluorescence signal from cells was recorded up to 12 days and plotted as fold change from day 1 for 1000 (FIG. 3A), 2000 (FIG. 3B), and 5000 (FIG. 3C) cell seeding densities. Representative longitudinal imaging for spheroids with 2000 seeded cells are shown (FIG. 3D). Next, fold change in OVCAR8-DsRed2 fluorescence was divided by fold change in NCI / ADR- RES-EGFP fluorescence to get the cell growth ratio (FIG. 3E). Total killing controls (5% bleach) were included, and viability is plotted as a function of fluorescence (FIG. 3F) and CellTiter-Glo® Cell Viability Assay (FIG. 3G). Representative images of total killing controlsare shown (FIG. 3H). Scale bar = 1000 m. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

[0019] FIG. 4A - 4G. Spheroid toxicity analysis of NP-Tal at varying doses. Spheroids composed of OVCAR8-DsRed2 cells and NCEADR-RES-EGFP cells were treated with NP- Tal at varying doses three days after seeding. Fluorescence was recorded up to 12 days, and intensity values for each cell line were normalized to the untreated spheroids to calculate cell viability. Longitudinal viability for parental and subline cells are shown from days 1-12 for NP-Tal doses at 0.01 pM (FIG. 4A), 0.04 pM (FIG. 4B), 0.11 pM (FIG. 4C), 0.33 pM (FIG. 4D) 1 pM (FIG. 4E), and 3 pM (FIG. 4F). Representative images of spheroids on day 12 at each treatment dose are shown (FIG. 4G). Scale bar = 1000 pm. * p < 0.05; ** p < 0.01; *** p < 0.001; ****p < 0.0001.

[0020] FIG. 5 A - 5 J. Treatment of 3D cocultures with PIC -NP-Tal. Spheroid cocultures of OVCAR8-DsRed2 and NCI / ADR-RES-EGFP cells were treated with PIC-NP-Tal or relevant controls three days after seeding. Light-activation was performed at 0 (dark control), 20, or 50 J / cm2. Viability analysis using the CellTiter-Glo® Cell Viability Assay was performed, where luminescence values were normalized to the no treatment (NT) 0 J / cm2control (FIG. 5A). Normalized luminescence is further analyzed within light doses for 0 (FIG. 5B), 20 (FIG. 5C), and 50 J / cm2(FIG. 5D). Fluorescence-based viability of each cell line, normalized to untreated spheroids, is shown at each light dose for NP-Tal (FIG. 5E), BPD (FIG. 5F), PIC (FIG. 5G), BPD+NP-Tal (FIG. 5H), PIC+NP-Tal (FIG. 51), and PIC-NP-Tal (FIG. 5J). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

[0021] FIG. 6A - 6B. Stability of nanoparticles with varying initial talazoparib amounts. Polymeric nanoparticles were prepared with 10.7 mg PLGA-PEG-COOH and 0, 0.107, 0.535, or 1.07 mg of talazoparib. Particle size (FIG. 6A) and Pdl (FIG. 6B) were tracked longitudinally for up to 24 weeks.

[0022] FIG. 7 A - 7B. Stability of nanoparticles with varying initial PLGA-PEG-COOH amounts. Polymeric nanoparticles were prepared with 0.535 mg of talazoparib and varied amounts of PLGA-PEG-COOH from 10.7 to 85.6 mg. Particle size (FIG. 7A) and Pdl (FIG. 7B) were tracked longitudinally for up to 24 weeks.

[0023] FIG. 8A - 8B Stability of nanoparticles with varying PLGA-PEG-DBCO / total polymer percentages. Polymeric nanoparticles were prepared with 0.535 mg of talazoparib and 42.8 total mg polymer. The polymer component was either PLGA-PEG-COOH, PLGA-PEG-DBCO, or a mixture. Particle size (FIG. 8A) and Pdl (FIG. 8B) were tracked longitudinally for up to 24 weeks.

[0024] FIG. 9A - 9B. Stability of PIC -conjugated nanoparticles. Polymeric nanoparticles were functionalized with PIC to establish PIC-NP and PIC-NP-Tal formulations. Both formulations were tracked for 12 weeks and particle size and Pdl were recorded (FIG 9 A and FIG 9B).

[0025] FIG. 10A - 10D. Longitudinal spheroid viability and growth tracking. Coculture spheroids were treated with NP-Tal up to 3 pM and imaged on days 1, 2, 4, 6, 8, 10, and 12. Fluorescence values were normalized to untreated cells on each respective day to quantify viability for OVCAR8-DsRed2 cells (FIG. 10A) and NCI / ADR-RES-EGFP cells (FIG. 10B). Growth dynamic of the parental cells (FIG. 10C) and subline (FIG. 10D) are calculated as the fold-change in RFU relative to day 1.

[0026] FIG. 11A - 11B. Day 12 spheroid viability curves. On day 12, spheroids treated with varying doses of NP-Tal were characterized for viability based on fluorescence of each cell line (FIG. 11 A) and luminescence in the CellTiter-Glo® Cell Viability Assay (FIG. 1 IB).

[0027] FIG. 12. Synthesis of Anti-PD-Ll-BPD (PIC).

[0028] FIG. 13. Anti-PD-Ll-BPD (PIC) absorbance and fluorescence. BPD conjugated to PD-L1 maintains the characteristic absorbance curve and fluorescence emission curve of free BPD.

[0029] FIG. 14. Anti-PD-Ll-BPD purity. PICs can be synthesized at a high purity of above 90% with minimal free BPD after purification shown via fluorescence imaging. PICs maintain a similar molecular weight to free antibody (-145 kDa), with slightly higher molecular weight fractions due to PEGylation.

[0030] Particular non-limiting embodiments of the present invention will now be described with reference to accompanying drawings.

[0031] DESCRIPTION

[0032] Delivery of poly (ADP-ribose) polymerase (PARP) inhibitor therapeutics can be difficult. The inventors surprisingly discovered that PARPi may be delivered via a nanoparticle particle composition disclosed herein for cancer treatment.

[0033] Combinational therapeutic strategies are a cornerstone in cancer therapeutics that may be leveraged to enable dose reductions of the individual therapies while maximizing anti-cancer effects. The inventors also surprisingly discovered that PARPi nanoparticleparticle composition disclosed herein for cancer treatment may be co-administered with photodynamic therapy (PDT). The PARPi nanoparticle particle composition may be modified to include an antibody conjugate (i.e., antibody-photosensitizer conjugate a / k / a photoimmunoconjugate) to facilitate photodynamic therapy (PDT).

[0034] A novel nanoplatform (an exemplary embodiment disclosed herein) was successfully engineered for ovarian cancer-targeted codelivery of talazoparib with photodynamic therapy (PDT).

[0035] PDT involves the light-activation of photosensitive dyes (photosensitizers) resulting in the generation of reactive molecular species which can induce direct cytotoxicity and modulate biological processes. Prior work has established harmonization between PDT and PARPi as an anti-cancer combination regimen for applications in ovarian, gastric, pancreatic, and skin cancers. Tanaka et al. found that talaporfm-mediated PDT enhanced P ARP -trapping capabilities of olaparib; and their combination significantly suppressed gastric tumor growth in a xenograft murine model. Lei et al. codelivered chlorine and olaparib to pancreatic cancer cells and demonstrated that their combination enhanced cytotoxicity, reactive oxygen species generation, and DNA damage. It was previously demonstrated that olaparib in combination with PDT using benzoporphyrin derivative (BPD) effectively reduced survival and clonogenicity of a coculture system of chemo-sensitive and chemo-resistant ovarian cancer cells. In the same study, it was further demonstrated that a lipidated photosensitizer formulation reduced selective survival advantage of the chemoresistant cells, effectively redirecting cancer evolution dynamics. This exemplifies the potential of nanoengineered combination therapies for overcoming critical barriers to clinical translation such as multidrug resistance. This is particularly relevant for PARP inhibitors, which have been shown in preclinical studies to induce acquired drug resistance through overexpression of multidrug resistance protein 1 (MDR1, P-gp, ABCB1). Rottenberg et al. compared olaparib-sensitive and olaparib-resistant tumors by quantifying the abcbla / b genes that encode for murine P-gp and found up to 85-fold increase in over 70% of resistant tumors compared to those sensitive. They further demonstrated that olaparib resistance could be reversed by the addition of a P-gp inhibitor, tariquidar. In another study, Oplustilova et al. showed, using a proliferation assay, that the P-gp inhibitor verapamil sensitized HCT116 colon cancer cells to PARP inhibitor KU 58948.

[0036] As discussed in more details herein, nanoengineering approaches are leveraged for encapsulation of talazoparib in polymeric nanoparticles (NP-Tal). NP-Tal is surface-decorated with antibody-photosensitizer conjugates (photoimmunoconjugates, PIC) using click chemistry for dual functionalization with cancer-targeting capabilities and PDT (PIC- NP-Tal). Optimal synthesis parameters are established to determine the masses of polymer and talazoparib added to the synthesis, as well as PIC-to-nanoparticle ratio. In parallel, a three-dimensional coculture model of fluorescently labelled ovarian cancer cells is developed to examine evolution of multi-drug resistance. The model is comprised of the parental OVCAR8-DsRed2 cells grown with their chemo-resistant P-gp overexpressing subline, NCI / ADR-RES-EGFP. This model enables fluorescence-based longitudinal viability tracking of each cell line in response to treatment, potentiating precise dose optimization. Results demonstrate that low dose NP-Tal (0.01 pM) trends towards selection of the drug-resistant populations by killing the parental OVCAR8-DsRed2 but sparing the NCI / ADR-RES-EGFP subline. In contrast, higher doses of NP-Tal (> 0.01 pM) kill both cell lines to similar degrees. Next, the combination of PIC and NP-Tal is compared to the conjugated PIC-NP-Tal to evaluate the role of conjugation on therapeutic effect. Results demonstrate potent combination effects of PIC and NP-Tal when mixed, but less potent effects when conjugated together. Additionally, treatment with PIC, BPD + NP-Tal, PIC + NP-Tal, and PIC-NP-Tal demonstrated selection pressures for the chemo-resistant subline, whereas NP-Tal alone and BPD alone kill both cell lines to equivalent degrees across all light doses tested. Results from this work provide fundamental implications for the combination of photoimmunotherapy (PIT) and PARP inhibition in the context of drugresistant ovarian cancer.

[0037] Within the last decade, poly(ADP-ribose) polymerase inhibitors (PARPi) have emerged in the clinic as an effective treatment for numerous malignancies. Preclinical data have demonstrated powerful combination effects of PARPi paired with photodynamic therapy (PDT), which involves light-activation of specialized dyes (photosensitizers) to stimulate cancer cell death through reactive oxygen species generation.

[0038] The present disclosure relates to a new PARP inhibitor formulation designed to mitigate side effects through the addition of cancer-targeting functionalization, while further enabling PARP inhibitor dose-reduction through combination treatment with photodynamic therapy. Moreover, one or more embodiments of the present disclosure can be designed to specifically target cancer cells while co-delivering photodynamic therapy for boosted anticancer action.

[0039] At least one embodiment of the present disclosure provides a method that includes dissolving PARP inhibitor and polymer in a water-miscible solvent, such as acetone, then adding to surfactant-containing water. The mixture is sonicated, and the acetone is evaporated, leaving behind nanoparticles that are subsequently filtered and concentrated. The nanoparticle size and PARP inhibitor loading are tunable based on initial polymer concentration. Photoimmunotherapy- functionalization is achieved by conjugating antibodyphotosensitizer conjugates (photoimmunoconjugates) to the nanoparticle surface. Photoimmunoconjugates are prepared using well-established EDC-NHS chemistry methods, and they are conjugated to PARP-loaded nanoparticles using copper-free click chemistry. The number of photoimmunoconjugates per nanoparticle is tunable from -30-300.

[0040] Embodiments of the present disclosure can provide photoimmunotherapy-enabled PARP inhibitor nanoparticles and novel synthesis procedure(s) that combine several well- established methods (nanoprecipitation, EDC-NHS chemistry, copper-free click chemistry). Embodiments of the present disclosure may be used in one or more of the following: Photodynamic therapy of cancer, PARP inhibition treatment of cancer, targeted treatment of cancer. Cancer targets may include (but are not limited to) gynecologic cancer, pancreatic cancer, prostate cancer, brain cancer, liver cancer, gastrointestinal cancer, head and neck cancer, skin cancer, soft tissue sarcoma, eye cancer, lung cancer, kidney cancer, esophageal cancer, peritoneal carcinoma, colorectal cancer, stomach cancer, peritoneal carcinomatosis, ovarian cancer, oral cancer, mesothelioma, and breast cancer.

[0041] In this report, the most potent clinical PARP inhibitor, talazoparib, is loaded into the core of a polymeric nanoparticle (NP-Tal), which is interfaced with antibody-photosensitizer conjugates (photoimmunoconjugates, PICs) to form PIC-NP-Tal. In parallel, a new 3D fluorescent co-culture model is developed using the parental OVCAR-8-DsRed2 and the chemoresistant subline, NCI / ADR-RES-EGFP. This model enables quantification of trends in the evolutionary dynamics of acquired chemoresistance in response to various treatment regimes. Results reveal that at a low dosage (0.01 pM), NP-Tal kills the parental cells while sparing the chemoresistant subline, thereby driving chemoresistance.

[0042] Next, PIC-NP-Tal and relevant controls are evaluated in the 3D coculture model at multiple irradiation doses to characterize effects on total spheroid ablation and relative changes in parental and subline cell population dynamics. Total spheroid ablation data shows potent combination effects when PIC and NP-Tal are co-administered, but decreased efficacy with the conjugated formulation (PIC-NP-Tal). Analysis of cell population dynamics reveal thatPIC, BPD+NP-Tal, PIC+NP-Tal, and PIC-NP-Tal demonstrate selection pressures towards chemoresistance. This invention provides key insights into manufacturing parameters for PARPi-loaded nanoparticles, as well as the potential role of combination therapies in the context of acquired drug resistance.

[0043]

[0044] Definitions

[0045] For the purposes of promoting an understanding of the principles of the invention, reference will now be made to certain embodiments and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, and alterations and modifications in the illustrated invention, and further applications of the principles of the invention as illustrated therein are herein contemplated as would normally occur to one skilled in the art to which the invention relates.

[0046] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0047] For the purpose of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with the usage of that word in any other document, including any document incorporated herein by reference, the definition set forth below shall always control for purposes of interpreting this specification and its associated claims unless a contrary meaning is clearly intended (for example in the document where the term is originally used).

[0048] The use of “or” means “and / or” unless stated otherwise.

[0049] The use of “a” or “an” herein means “one or more” unless stated otherwise or where the use of “one or more” is clearly inappropriate.

[0050] The use of “comprise,” “comprises,” “comprising,” “include,” “includes,” and “including” are interchangeable and not intended to be limiting. Furthermore, where the description of one or more embodiments uses the term “comprising,” those skilled in the art would understand that, in some specific instances, the embodiment or embodiments can be alternatively described using the language “consisting essentially of’ and / or “consisting of.”

[0051] As used herein, the term “about” refers to a ±10% variation from the nominal value. It is to be understood that such a variation is always included in any given value provided herein, whether or not it is specifically referred to.

[0052] A “therapeutically effective amount” refers to the amount of the photo- immunoconjugate formulation sufficient to elicit a desired biological response in a subject, e.g., such as an amount sufficient to kill, reduce or stabilize cells associated with a disease or condition and / or sufficient to reduce symptoms associated with such disease or condition. Actual dosage levels of the active ingredient(s) in the disclosed formulations may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration. The formulations and compositions of the present invention may be administered by any suitable route and mode (e.g., parenteral, injected, topical, oral, intranasal, etc.).

[0053] Numeric ranges recited within the specification are inclusive of the numbers defining the range and include each integer within the defined range. Throughout this disclosure, various aspects of this disclosure are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges, fractions, and individual numerical values within that range. For example, a description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6, and decimals and fractions, for example, 1.2, 3.8, P , and 4% This applies regardless of the breadth of the range.

[0054] As used herein, a “photosensitizer” or “photoreactive agent” is a compound or composition that is useful in photodynamic therapy in that it absorbs electromagnetic radiation and emits energy sufficient to exert a therapeutic effect, e.g., the impairment or destruction of unwanted cells or tissue, or sufficient to be detected in diagnostic applications. Photodynamic therapy according to the invention can be performed using any of a number of photoactive compounds. For example, the photosensitizer can be any chemical compound that collects in one or more types of selected target tissues and, when exposed to the light of a particular wavelength, absorbs the light and induces impairmentor destruction of the target tissues. Virtually any chemical compound that homes to a selected target and absorbs light may be used in this invention. Preferably, the photosensitizer is nontoxic to the patient to which it is administered and is capable of being formulated in a nontoxic composition. The photosensitizer is also preferably nontoxic in its photodegraded form. Ideal photosensitizers are characterized by a lack of toxicity to cells in the absence of the photochemical effect and are readily cleared from non-target tissues.

[0055] “Theoretical loading capacity” and “loading capacity” herein refer to the amount of photosensitizers loaded per unit weight of the nanoparti cle(s), with the former encompassing the calculated loading capacity and the former the empirical loading capacity. The nanoparticles described herein may have a loading capacity and / or theoretical loading capacity of up to 100% by weight, including 50 wt.%, 60 wt.%, 70 wt%, 80 wt.%, 90 wt.% and all integers included within these ranges. For example, the loading capacity of the nanoparticle compositions described herein is up to 100% by weight (i.e., 100 wt.%) or is 100 wt.% with, for example, 706,000+38,000 verteporfm molecules per nanoparticle. A 100% loading capacity beneficially permits carrier-free and / or lipid-free compositions and delivery of the same. In comparison, most of the existing nanomedicines possess the drawback of low drug-loading capacity (generally less than 10%) associated with more carrier materials.

[0056] The term “nanoparticle carrier” refers to small transport agents that can be modified in terms of size, charge, and shape to carry therapeutic agents to specific tissues.

[0057] The term “polymeric nanoparticle” refers to organic-based nanoparticles that are solid colloidal particle systems in which drug material is either entrapped, encapsulated and dissolved in a polymeric matrix or adsorbed onto matrix.

[0058] The term “water-miscible” refers to a liquid that can mix fully mix with water and does not leave distinct layers between them in solution.

[0059] The term “surfactant” refers to a substance that is added to a liquid to reduce the surface tension and increase the spreading and wetting properties.

[0060] The abbreviation “BPD” means benzoporphyrin derivative.

[0061] The abbreviation “Cet” means cetuximab.

[0062] The abbreviation “EGFR” means epidermal growth factor receptor.

[0063] The abbreviation “EMA’ means European Medicines Agency.

[0064] The abbreviation “FDA” means United States Food and Drug Administration.

[0065] The abbreviation “NAD+” means nicotinamide adenine dinucleotide.

[0066] The abbreviation “NP-Tal” means talazoparib-loaded nanoparticle.

[0067] The abbreviation “PARP” means poly(ADP-ribose) polymerase.

[0068] The abbreviation “PARPi” means poly(ADP-ribose) polymerase inhibitor.

[0069] The abbreviation “PIC” means photoimmunoconjugate.

[0070] The abbreviation “PIC -NP-Tal” means photoimmunoconjugate-functionalized talazoparib-loaded nanoparticle.

[0071] The abbreviation “SSB” means single strand break.

[0072] The abbreviation “XRCC1” means X-ray repair cross-complementing protein 1.

[0073] The methods, systems, apparatuses, and compositions disclosed herein may comprise, consist essentially of, or consist of the components and ingredients described herein as well as other ingredients not described herein. As used herein, “consisting essentially of’ means that the methods, systems, apparatuses, and compositions may include additional steps, components, or ingredients, but only if the additional steps, components, or ingredients do not materially alter the basic and novel characteristics of the claimed methods, systems, apparatuses, and compositions.

[0074] LIST OF EXPEMLARYEMBODIMENTS1. A poly (ADP-ribose) polymerase (PARP) inhibitor nanoparticle composition, said composition comprising a PARP inhibitor and a polymer, wherein said composition is in the form of a nanoparticle particle.2. The composition of embodiment 1, wherein said nanoparticle has a size of about 50 nm to about 500 nm.3. The composition of any of the preceding embodiments, wherein said PARP inhibitor is talazoparib, olaparib, rucaparib, veliparib, and niraparib, or a combination thereof.4. The composition of any of the preceding embodiments, wherein said polymer is polylactic acid (PLA), poly(lactide-co-glycolide) (PLGA), poly(lactide-co-glycolide)- block-poly(ethylene glycol) (PLGA-PEG), poly(lactide-co-glycolide)-block- poly(ethylene glycol)-carboxylic acid (PLGA-PEG-COOH), or poly(lactide-co- glycolide)-block-poly(ethylene glycol)-dibenzocyclooctyne-amine (PLGA-PEG- DBCO).5. The composition of embodiment 4, wherein said polymer is poly(lactide-co-glycolide)- block-poly(ethylene glycol)-carboxylic acid (PLGA-PEG-COOH), or poly(lactide-co-glycolide)-block-poly(ethylene glycol)-dibenzocyclooctyne-amine (PLGA-PEG- DBCO).6. The composition of any of the preceding embodiments, wherein said composition further comprises a photosensitizer.7. The composition of embodiment 6, wherein said photosensitizer is coupled to the surface of said nanoparticle composition via an antibody conjugate (i.e., antibodyphotosensitizer conjugate a / k / a photoimmunoconjugate).8. The composition of embodiments 6-7, wherein said photosensitizer is a benzoporphyrin derivative (BPD) photosensitizer, a chlorin-based photosensitizer, a porphyrin-based photosensitizer, a bacteriochlorin-based photosensitizer, a phthalocyanine-based photosensitizer, a ruthenium-based photosensitizer, or a fluorescence imaging agent.9. A method of preparing a PARP inhibitor nanoparticle composition, said method comprising: dissolving a PARP inhibitor and a polymer in a water-miscible solvent (e.g., acetone) to obtain a PARP inhibitor-polymer solution; and adding to surfactant-containing water to obtain a mixture and sonicating said mixture; removing said solvent (e.g., evaporation) to obtain said PARP inhibitor -polymer nanoparticle.10. The method of embodiment 9, wherein said PARP inhibitor has a concentration of about 0 mg / mL to about 100 mg / mL.11. The method of embodiment 9, wherein said polymer is chosen from PLA, PLGA, PLGA-PEG, PLGA-PEG-COOH and PLGA-PEG-DBCO, or a combination thereof.12. The method of embodiment 11, wherein said polymer is a combination of PLA, PLGA, PLGA-PEG, PLGA-PEG-COOH and PLGA-PEG-DBCO.13. The method of embodiment 12, wherein the mass of said PLGA-PEG-DBCO to PLGA-PEG-COOH ratio is from 0: 100 to 100:0.14. The method of embodiment 9, wherein said water-miscible solvent is added to water in the amount of about 0.1 %v / v to about 50 %v / v.15. The method of embodiment 14, wherein said water-miscible solvent is added to water in the amount of about 10% v / v.16. The method of embodiment 9, wherein said water-miscible solvent is acetone, ethanol, acetonitrile, anisole, chloroform, dichloro- methane, dimethylformamide, dimethylsulfoxide, ethyl acetate, or dioxane.17. The method of embodiment 9, wherein said water-miscible solvent is acetone.18. The method of embodiment 9, wherein said surfactant containing water comprises about 0.01% surfactant to about 10% surfactant.19. The method of embodiment 9, wherein said surfactant is a pluronics.20. The method of embodiment 19, wherein said pluronics is Pluronic F-68.21. The method of any of the preceding embodiments, further comprising adding an antibody-photosensitizer conjugate to a surface of said PARP inhibitor-polymer nanoparticle to obtain a conjugated nanoparticle.22. The method of embodiment 21, wherein the molar ratio of said antibody- photosensitizer conjugate to said PARP inhibitor-polymer nanoparticle is about 1 : 1 to about 1000:1.23. The method of embodiment 21, wherein said antibody-photosensitizer conjugate and said PARP inhibitor-polymer nanoparticle are mixed overnight.24. The method of embodiment 23, wherein said antibody-photosensitizer conjugate and said PARP inhibitor-polymer nanoparticle are mixed for about 0.1 hours to about 24 hours.25. The method of embodiment 21, wherein said antibody-photosensitizer conjugate is added to said surface of said PARP-polymer nanoparticle using copper-free click chemistry.26. The method of embodiment 21, wherein said conjugated nanoparticle is purified by size exclusion chromatography.27. A method for the treating cancer, said method comprising administering a nanoparticle composition of any of the preceding embodiments and optionally photosensitizer to a subject.28. A method for the treating cancer, said method comprising administering a nanoparticle composition of any of the preceding embodiments and photosensitizer of any of the preceding embodiments to a subject.29. The method of embodiment 27, wherein said composition and said photosensitizer are co-administered separately.30. The method of embodiment 27, wherein said photosensitizer is coupled to the surface of said nanoparticle composition via an antibody conjugate.31. The method of any of the preceding embodiments, said method further comprising subjecting said subject to photodynamic therapy or photoimmunotherapy.32. The method of embodiment 27, wherein said cancer is an epidermal growth factor receptor (EGFR) expressing cancer.33. The method of embodiment 27, wherein said cancer is a programmed death-ligand 1 (PD-L1) expressing cancer.34. The method of embodiment 27, wherein said cancer is selected from the group consisting of gynecologic cancer, pancreatic cancer, prostate cancer, brain cancer, liver cancer, gastrointestinal cancer, head and neck cancer, skin cancer, soft tissue sarcoma, eye cancer, lung cancer, kidney cancer, esophageal cancer, peritoneal carcinoma, colorectal cancer, stomach cancer, peritoneal carcinomatosis, ovarian cancer, oral cancer, mesothelioma, and breast cancer.35. The method of any of the preceding embodiments, wherein said composition are administered via a route chosen from oral administration, intravenous, intra-arterial, intratumoral, intravesical, intramuscular, subcutaneous, and intraperitoneal injections.36. The method of any of the preceding embodiments, wherein said photosensitizer is administered via a route chosen from oral administration, intravenous, intra-arterial, intratumoral, intravesical, intramuscular, subcutaneous, and intraperitoneal injections.37. The method of embodiment 27, wherein said photosensitizer coupled to the surface of said nanoparticle composition via an antibody conjugate is administered via a route chosen from oral administration, intravenous, intra-arterial, intratumoral, intravesical, intramuscular, subcutaneous, and intraperitoneal injections.

[0075] EXAMPLES

[0076] The following examples are provided solely to illustrate the present invention and are not intended to limit the scope of the invention, described herein.

[0077] Example 1. Synthesis of Photoimmunoconjugates

[0078] PIC synthesis was performed as previously described. First, 10 kDa methoxy PEG succinimidyl carboxymethyl ester (mPEG-NHS; JenKem Technology) was added dropwise to Cetuximab at a 3 : 1 molar ratio and reacted overnight under continuous stirring. Next, BPD N- hydroxysuccinimidyl ester (BPD-NHS) and azide-PEG4-7V-hydroxysuccinimidyl ester (azide-PEG-NHS; Thermo Scientific) were added to the reaction to a final ratio of 9 and 2.5 moles per 1 mole Cetuximab, respectively. After another 20 hours of stirring, the mixture was purified using a 30 kDa Zeba spin desalting column (7 kDa MWCO; Thermo Scientific) and concentrated using an Amicon centrifugal filter unit (30 kDa MWCO, Millipore Sigma). Final Cetuximab concentration was determined by Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific), and final BPD concentration was determined by UV-vis spectroscopy.

[0079] Example 2. Synthesis of PIC -Functionalized Polymeric Nanoparticles

[0080] For talazoparib-loaded nanoparticles (NP-Tal), synthesis parameters were initially varied for protocol optimization (Table 1). PLGA-PEG-COOH and PLGA-PEG-DBCO were obtained from PolySciTech, and talazoparib (Tai) was obtained from MedChemExpress. Polymer was first co-dissolved with talazoparib in 1 mL of acetone, then added to 10 mL of ultrapure water (Invitrogen) containing 0.1% Pluronic F-68 (Gibco). The solution was sonicated with a probe sonicator for 3 minutes and acetone was evaporated at room temperature for 4-6 hours under continuous stirring at 400 rpm. The obtained NP-Tal were filtered through 0.22 pm syringe filter units (Millipore) and concentrated in an Amicon centrifugal filter unit (30 kDa MWCO, Millipore Sigma). Next, PIC was conjugated to the DBCO-containing nanoparticles through copper-free click chemistry. For conjugation, PIC and nanoparticles were mixed overnight at volume ratios of 0.5: 1, 1 :1, 2: 1, and 3: 1, then purified via Sepharose CL-4B size exclusion chromatography.

[0081] Table 1. Varying Parameters in Nanoparticle Formulation

[0082] Example 3. Photophysical and Photochemical Nanoparticle Characterization

[0083] Talazoparib concentration was determined using a fluorescence-based standard curve (ex / em; 312 / 416 nm, Synergy neo2, Biotek). BPD concentration was calculated similarly (ex / em; 435 / 700nm). Loading capacity (%) was calculated as the mass of polymer divided by the mass of loaded talazoparib. The talazoparib encapsulation efficiency (%) was calculated as the ratio of nanoparticle-loaded talazoparib to the initial talazoparib added to the nanoparticle synthesis reaction. Talazoparib retention (%) was calculated as the ratio of talazoparib afterand before PIC conjugation. PIC conjugation efficiency (%) was determined by calculating the ratio of BPD loaded onto the nanoparticle to the initial BPD added to the conjugation reaction. PIC per nanoparticle was calculated by first determining molecules of PIC using Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific), then dividing by the number of nanoparticles as determined by NanoSight LM10 (Malvern Instruments). Talazoparib per nanoparticle was calculated as molecules of talazoparib divided by number of nanoparticles. Nanoparticle size, poly dispersity index, and zeta potential were determined using the Nanobrook Omni (Brookhaven Instruments). To quantify photoactivity, compounds were dissolved in PBS or DMSO, then fluorescence emission was collected upon light-activation at 435 nm. Maximum fluorescence emission in PBS was divided by maximum fluorescence emission in DMSO for photoactivity values. Singlet oxygen generation was determined using the Singlet Oxygen Sensor Green (SOSG) probe (Invitrogen). Selectivity and uptake studies of PIC versus PIC- NP-Tal were performed with 0VCAR8 (EGFR+) and J774 (EGFR-) cells. First, 300,000 cells were plated and incubated overnight. Next, dishes were treated with 1 pM PIC or PIC-NP-Tal for 30 minutes. Cells were lysed using radioimmunoprecipitation assay buffer, then BPD fluorescence was measured at ex / em 435 / 700 nm.Example 4. 3D Ovarian Cancer Coculture System Development and Treatment Regimen

[0084] High grade serous ovarian cancer cell lines OVCAR8-DsRed2 and NCVADR-RES- EGFP were obtained courtesy of Dr. Michael M. Gottesman (National Cancer Institute, National Institutes of Health). Both cell lines were cultured in RPMI-1640 medium (Coming) supplemented with 10% fetal bovine serum (Gibco), 100 U / mL penicillin and 100 pg / mL streptomycin (Coming). Every four passages, media was supplemented with G418 (Invitrogen) at 500 pg / mL (OVCAR8-DsRed2) or 200 pg / mL (NCI-ADR-RES-EGFP). The growth dynamics of these cell lines on 2D substrates was described previously by our group. 3D spheroidal cocultures were generated by plating equal numbers of OVCAR8-DsRed2 and NCI / ADR-RES-EGFP to a final cell number of 1,000, 2,000, or 5,000 cells per well in ultra- low-attachment, round bottom 96 well plates (PerkinElmer). The Lionheart FX Automated Microscope (Biotek) was used for imaging 4 hours after plating, 24 hours after plating, and then every two days up to day 12. For treatment evaluation, 2,000 cells (1,000 of each cell line) were treated on day 4 for 24 hours prior to light activation (690 nm, Modulight, Inc.) on day 5. Longitudinal imaging was conducted as described above, and final cell viability analysis was conducted on day 12 using the CellTiter-Glo® Cell Viability Assay (Promega). Total killingcontrols were achieved by treating spheroids on day 12 with 5% bleach for four hours prior to viability analysis.

[0085] Example 5. Statistical Analysis

[0086] GraphPad Prism version 9.0.2 was used for statistical analysis. All data shown were collected at least in triplicate and plotted as mean ± standard error of the mean. Details regarding statistical testing are elaborated in figure captions, and statistical significance was determined as P < 0.05.

[0087] Example 6. Development of Talazoparib-Loaded Polymeric Nanoparticles

[0088] NP-Tal were prepared by nanoprecipitation methods, where acetone and ultrapure water were used for the organic and aqueous phase, respectively (Figure 1 A). A representative TEM image of the NP-Tal is shown in Figure IB. The development of nanoparticle synthesis optimization began with varying the initial amount of talazoparib added to the reaction (Figure 1 C-G; Figure 6). For these studies, the amount of polymer (PLGA-PEG-COOH) added to the reaction was fixed at 10.7 mg. Tested masses of talazoparib included 0, 0.107, 0.535, 1.070, and 2.675 mg. The loading capacity was first measured (Figure 1C). Loading capacity initially increases, then plateaus, where a maximum is reached at 0.535 mg. This is further shown by encapsulation efficiency data (Figure ID), calculated as the percent of initially added talazoparib that is successfully encapsulated in the nanoparticles. Encapsulation efficiency is consistently at -3% when 0.107 and 0.535 mg are added (p < 0.01), then decreases at higher values. NP-Tal are ~15 nm larger than empty nanoparticles (Figure ID), and sizes remain stable across formulations for up to 24 weeks (Figure 6A). Pdl of all formulations was initially below 0.16 (Figure IF) and remained stable at or below 0.21 for up to 24 weeks (Figure 6B). Zeta potentials (Figure 1G) remain relatively consistent across batches. For subsequent reactions, 0.535 mg was set as the talazoparib mass due to maximum loading being reached.

[0089] Next, the mass of PLGA-PEG-COOH added in the nanoparticle synthesis reaction was varied (Figure 1H-1L). The initial mass of talazoparib was fixed at 0.535 mg, and polymer was added at 10.7, 21.4, 42.8 or 85.6 mg. No significant changes in loading capacity were calculated (Figure 1H), encapsulation efficiency increased with increasing polymer mass (Figure II), and size increased significantly for nanoparticles made with 42.8 and 85.6 mg polymer (Figure 1 J). Up to 24 weeks, size of all groups remained stable (Figure 7A). The poly dispersity index remained relatively consistent (-0.16) at 10.7, 21.4, 42.8 mg polymer but increased significantly at 85.6 mg polymer to 0.22 (p < 0.01) (Figure IK). Pdl for all groups remained consistent for up to 24 weeks (Figure 7B). Zeta potential remained relatively consistent acrossformulations (Figure IL). Based on these results, 42.8 mg polymer was selected as the optimal mass of polymer due to the significant increase in Pdl at 85.6 mg.

[0090] In order to later ‘click’ azide-functionalized photoimmunoconjugates (PIC) onto the nanoparticle, PLGA-PEG-DBCO was incorporated into the formulation. PLGA-PEG-DBCO was mixed with PLGA-PEG-COOH to a final total polymer mass of 42.8 mg, and the relative mass of PLGA-PEG-DBCO was varied from 0, 25, 50, and 100% (Figure IM- IQ). The talazoparib loading capacity and encapsulation efficiency remained relatively consistent with increasing mass of PLGA-PEG-DBCO (Figure IM - IN). In contrast, size, Pdl, and zeta potential increased significantly when nanoparticles were prepared with 100% PLGA-PEG- DBCO (Figure 10 - IQ). Size and Pdl remained stable across 24 weeks for groups where the mass percent of PLGA-PEG-DBCO was below 100% (Figure 8 A- 8B). However, for 100% PLGA-PEG-DBCO nanoparticles, after 24 weeks, size decreased from -300 nm to below 200 nm, and Pdl decreased from -0.3 to 0.22. Due to the instability and high Pdl of 100% PLGA- PEG-DBCO, the 50% PLGA-PEG-DBCO condition was selected for subsequent experiments.

[0091] Example 7. Optimization and Characterization of PIC-Conjugated Nanoparticles

[0092] Azide-functionalized PICs composed of Cetuximab and benzoporphyrin derivative (BPD) were ‘clicked’ onto the surface of DBCO-functionalized nanoparticles (Figure 2 A - 2B). PICs were first prepared using carbodiimide chemistry at a 4: 1 final BPD:Cetuximab ratio. Next, 100 pL of nanoparticles were reacted overnight with PIC at varying volumes (50, 100, 200, 300 pL) resulting in volume ratios of 0.5: 1, 1 : 1. 2: 1, and 3: 1 (PIC:NP). PIC-conjugated nanoparticles (PIC-NP) were purified by size exclusion chromatography and characterized for size, PIC conjugation efficiency (based on BPD concentration), and PIC per nanoparticle (Figure 2C - 2E). Dynamic light scattering data revealed that PIC conjugation to nanoparticles increased particle diameter by -10 nm (Figure 2C). PIC conjugation efficiency (amount of photosensitizer conjugated to the nanoparticle relative to the amount added to the synthesis reaction) increased with higher PIC:NP reaction volume ratios, reaching a plateau at the 2: 1 volume ratio around -32% (Figure 2D). Next, the number of PICs per nanoparticle was calculated, revealing a range from -30-330 PIC / NP at varying reaction volumes (Figure 2E). Due to the plateau in reaction efficiency occurring at a 2: 1 PIC:NP volume ratio, this condition was selected for subsequent studies. Stability of PIC-NP and PIC-NP-Tal in size and Pdl was confirmed for up to 12 weeks (Figure 9).

[0093] PIC-NP-Tal was next prepared and characterized (Figure 2F - 2K, Table 2). Absorbance spectra were recorded (Figure 2F - 2G), demonstrating that all BPD-containingagents (BPD, PIC, PIC-NP, PIC-NP-Tal) have the characteristic BPD absorbance peaks at -435 nm and -700 nm. Tal-containing formulations show characteristic absorbance peaks at -312 nm. Next, quenching in aqueous solution was evaluated by comparing absorbance at 690 nm in PBS (quenched) versus DMSO (unquenched) (Figure 2H). BPD, PIC, PIC-NP, and PIC- NP-Tal all exhibit quenching, shown as significant reductions (-20-50%) in absorbance in PBS compared to DMSO. Free BPD and PIC show low photoactivity (<7%) due to quenching in aqueous solution, whereas the photoactivity of PIC-NP and PIC-NP-Tal is significantly higher, at 42% and 33%, respectively (Figure 21). In Figure 2J, singlet oxygen generation based on SOSG fluorescence signal is shown. Compared to BPD, PIC-NP and PIC-NP-Tal show significantly higher fluorescence emission intensity (P < 0.01), representing elevated singlet oxygen yield.

[0094] EGFR-dependent uptake was next evaluated by treating EGFR-negative J774 cells and EGFR-positive OVCAR8 cells with PIC or PIC-NP-Tal for 30 minutes, then collecting cells and quantifying internalized BPD (Figure 2K). PIC uptake by OVCAR8 cells was significantly (P < 0.01) greater than PIC uptake by J774 cells with a 3.5-fold increase in photosensitizer uptake, demonstrating EGFR-enhanced uptake. For PIC-NP-Tal, uptake by OVCAR8 cells was over double that of J774 (P < 0.05).

[0095] Nanoparticle properties are summarized in Table 2. Empty nanoparticles (NP) are -140 nm, whereas talazoparib loaded nanoparticles (NP-Tal) are -126 nm. This difference in size is not statistically significant (P = 0.30), and upon PIC conjugation, both nanoparticles increase in size by 5-7 nm. Pdl for empty and talazoparib-loaded nanoparticles is around -0.18, whereas PIC conjugated nanoparticles have Pdl just under 0.20. For all formulations, zeta potential is consistently around -7 mV. Talazoparib loading into nanoparticles was determined as 8.5%, and molecules of talazoparib per nanoparticle were calculated as 5295.1 and 2104.4 for NP- Tal and PIC-NP-Tal, respectively. PIC conjugation efficiency is -30% for PIC-NP and PIC- NP-Tal and the number of PIC per nanoparticle is consistent between both formulations at -115 PIC / NP.

[0096] Talazoparib loading efficiency is defined as the moles of talazoparib loaded into the nanoparticle divided by the moles of talazoparib added to the nanoparticle synthesis reaction. # talazoparib per NP is defined as the molecules of talazoparib divided by the number of nanoparticles. PIC conjugation efficiency is defined as the moles of BPD conjugated to the nanoparticle divided by the moles of BPD added to the initial conjugation reaction. # PIC perNP is defined as the molecules of PIC (based on antibody) divided by the number of nanoparticles. Each datapoint is representative of at least four individual nanoparticle batches.

[0097] Table 2. Characterization of nanoparticle physical properties and drug loading.

[0098] Example 8. Development of 3 -Dimensional Ovarian Cancer Coculture Model

[0099] A 3D coculture model of a parental (OVCAR8-DsRed2) and a chemo-resistant subline (NCI / ADR-RES-EGFP) was developed by seeding 1,000, 2,000, or 5,000 cells at a 1 : 1 ratio in ultra-low attachment round bottom plates and tracking fluorescence over the course of 12 days (Figure 3A - 3C). Representative longitudinal images of spheroids with a 2000 cell seeding density are shown in Figure 3D. The parental OVCAR8-DsRed2 cells grew drastically faster than NCI / ADR-RES-EGFP at all seeding densities, reaching 48-, 58-, and 215-fold increases in RFU by day 12 in 5000, 2000, and 1000 cell seeding densities, respectively. NCI / ADR- RES-EGFP cells, in contrast reach 1-, 2-, and 6-fold increases in growth by day 12 for 5,000, 2,000, and 1,000 seeding density groups. The growth ratios of OVCAR8-DsRed2:NCI / ADR- RES-EGFP were calculated as the fold change in OVCAR8-DsRed2 RFU relative to day 1 divided by the fold change in NCI / ADR-RES-EGFP RFU relative to day 1 (Figure 3E). Across all starting seeding densities, this ratio remained relatively consistent over the course of the experiment, with day 12 values at 40, 25, and 41 at 1,000, 2,000, and 5,000 densities, respectively (P > 0.4). Fluorescence-based viability tracking was next validated by preparing a total killing control (5% bleach, 4 hours) and comparing fluorescence emission intensity values with an ATP -based cell viability assay (CellTiter-Glo® Cell Viability Assay) (Figure 3F - 3G). Fluorescence intensity of OVCAR8-DsRed2 and NCI / ADR-RES-EGFP decreasedsignificantly for total killing controls to 13% and 28%, respectively. In contrast, the ATP -based assay showed reductions in viability down to <1%. The residual fluorescence values for total killing controls are likely resulting from auto-fluorescent contributions. Representative images of total killing controls are shown in Figure 3H.

[0100] Example 9. Comparative Dosage Analysis ofNP-Tal in Ovarian Cancer 3D Cocultures

[0101] Next, treatment effect of NP-Tal in spheroids (2,000 cell seeding density) was evaluated using concentrations from 0.01 to 3 pM (Figure 4). On day 4, when spheroids were fully established, they were treated with varying doses of NP-Tal until day 12. Images were taken longitudinally, and the fluorescence emission intensity of each spheroid was normalized to the untreated spheroid on each respective day to determine viability (Figure 4A - 4F). Representative images of spheroids on day 12 are shown (Figure 4G). Results at the lower NP- Tal doses (0.01 - 0.11 pM) demonstrate a decrease in viability for the parental cell line whereas the resistant subline was spared (Figure 4A - 4C). On the other hand, higher doses (0.33 - 3 pM) killed both parental and subline cells to a similar degree (Figure 4D - 4F). Figure 10A - 10B shows fluorescence-based viability analysis of OVACR8-DsRed2 cells and NCI / ADR- RES-EGFP cells, revealing decreases in viability with increasing NP-Tal dosing. On day 12, 3 pM treatment resulted in -14% and -25% viability for OVACR8-DsRed2 cells and NCI / ADR- RES-EGFP cells, respectively. Next, growth curves were calculated based on changes in fluorescence relative to day 1 for each cell line (Figure 10C - 10D). Untreated cells show day 12 growth increases at 58-fold and 2-fold for OVACR8-DsRed2 cells and NCVADR-RES- EGFP cells, respectively. Increasing NP-Tal dosage caused decreasing fold changes in growth, with day 12 values at 8-fold and 0.6-fold for OVCAR8-DsRed2 cells and NCVADR-RES- EGFP cells, respectively, with 3 pM treatment. Dose-dependent effects of NP-Tal are shown in Figure 11 A - 1 IB using fluorescence-based and ATP-based viability assays. Fluorescencebased data shows a rightward shift of the NCI / ADR-RES-EGFP cells, representing increased resistance to NP-Tal relative to the parental OVCAR8-DsRed22 cell line.

[0102] Example 10. PIC-NP-Tal Treatment Outcomes in 3D Spheroid Cocultures

[0103] PIC-NP-Tal and monotherapy controls were next tested in the 3D coculture model (Figure 5). Luminescence-based viability analysis in Figure 5A shows that there are light-dose dependent effects of BPD, PIC, BPD mixed with NP-Tal (BPD+NP-Tal), and PIC mixed with NP-Tal (PIC+NP-Tal). In contrast, there were no significant light-dose dependent toxicities for the no treatment (NT), NP-Tal, and PIC-NP-Tal groups. Analysis of treatment groups within light doses is shown in Figures 5B - 5D. At all light doses, PIC-NP-Tal does not inducesignificant reductions in viability. In contrast, when PIC and NP-Tal are mixed as an unconjugated pair (PIC+NP-Tal), spheroid viability is reduced to 84% (P < 0.05), 53% (P < 0.0001), and 17% (P < 0.0001) at 0, 20, and 50 J / cm2. Notably, at 20 J / cm2, PIC+NP-Tal significantly outperforms PIC and NP-Tal alone. BPD alone, PIC alone, and BPD+NP-Tal caused significant reductions in viability at 20 and 50 J / cm2, and PIC+NP-Tal significantly outperformed PIC-NP-Tal at 20 and 50 J / cm2. Next, parental and subline fluorescence intensities were normalized to untreated spheroids and plotted in Figure 5E - 5 J. NP-Tal and BPD did not cause significant differences in viability between cell lines across all light doses, demonstrating a lack of selection pressures for either cell line. Interestingly, BPD+NP-Tal does select for chemoresistance, as determined by significantly higher viability of the NCI / ADR- RES-EGFP line compared to the OVCAR8-DsRed2 line at 0 and 20 J / cm2. PIC alone, PIC+NP-Tal, and PIC-NP-Tal induce selection pressures towards drug resistance at 20 and 50 J / cm2, but not at 0 J / cm2.

[0104] Example 11. Anti-PD-Ll and BPD photoimmunoconjugate (PIC)

[0105] It is now possible to prepare antibody-photosensitizer conjugates (photoimmunoconjugate s) that target programmed death-ligand 1 (PD-L1) expressing cancer. Anti-PD-Ll PIC is reproducible, with average BPD concentration of -150 pM, average antibody concentration of -9 mg / mL, and BPD: Ab ratio between 2 and 3 (Table 3)

[0106] Table 3. Representative Batch Parameters.

[0107] Example 12. Discussion

[0108] PARP inhibition has emerged in recent years as a powerhouse chemotherapy for numerous malignancies. In the clinic, PARP inhibitors are used to treat a growing list of indications that currently includes ovarian, breast, pancreatic, prostate, fallopian, and primary peritoneal cancers. Of the currently FDA-approved PARP inhibitors, studies show that talazoparib has the lowest IC50 and greatest PARP trapping capabilities. However, talazoparib is also the most toxic, with at least 300-fold lower maximum tolerated dose compared to theother clinically-prescribed PARP inhibitors. Nanoengineering approaches have emerged as a promising strategy to overcome this obstacle and strengthen PARP inhibition as an anti-cancer modality.

[0109] This nanoengineering approach is unique in that it combines talazoparib with photoimmunotherapy to achieve codelivery of PDT and PARP inhibition within a targeted formulation. Work by Spring et al. has demonstrated the capabilities of photoimmunotherapy for ovarian cancer treatment in vivo. They showed that anti-EGFR photoimmunoconjugates composed of Cetuximab and BPD could selectively accumulate in ovarian cancer metastases, enabling precise imaging and treatment. It was previously shown that photoimmunotherapy- functionalized nanoparticles promote enhanced photosensitizer delivery and possess combination-treatment capabilities through co-encapsulation of additional therapeutic entities. Additionally, the clinical relevance of photoimmunotherapy -based anti-cancer approaches has recently been elevated with the clinical use of Cetuximab-IR700 conjugates for the treatment of head and neck cancer in Japan.

[0110] The present study combines two clinically relevant modalities, photoimmunotherapy and PARP inhibition, in a targeted polymeric nanoparticle for the treatment of ovarian cancer spheroids. First, the nanoformulation was optimized through modulating various synthesis parameters including talazoparib mass, polymer mass, and ratio of two polymers (PLGA-PEG- COOH and PLGA-PEG-DBCO) (Table 1, Figure 1). The optimized formulation was then functionalized with photoimmunoconjugates via copper-free click chemistry for targeting and photoactivity capabilities (Figure 2). Consistent with previous work, the final formulation (PIC-NP-Tal) retained the 690 nm Q-band of BPD for light activation and showed superior singlet oxygen generation compared to free BPD. Like PIC, PIC-NP-Tal also demonstrated selectivity for EGFR-expressing cells. Importantly, uptake of PIC-NP-Tal by EGFR- expressing cells was greater than uptake of PIC alone by 45%, demonstrating our previously described “carrier effect” phenomenon in 2-dimensional cultures.

[0111] In parallel, a novel fluorescent 3D coculture system of the parental OVCAR8-DsRed2 cells and the drug resistant subline, NCI / ADR-RES-EGFP (Figure 3) was developed. In a previous study, these cell lines were cocultured on 2D substrate, leading to rapid domination of the parental subline. For example, after 7 days, the parental line outnumbered the subline by nearly 5-fold, and by 14 days this difference increased to ~20-fold. Similarly, in 3D growth conditions, the parental cell line rapidly outgrows the chemo-resistant subline, and this trend is consistent when cells are plated at varying seeding densities (1000, 2000, 5000 cells perwell). Spheroids with lower seeding densities showed greater increases in cellular fluorescence compared to spheroids plated at higher seeding densities, representative of greater spheroid growth (Figure 3A - 3C). For example, at the 1000 cell seeding density, the fluorescence emission intensity of OVCAR8-DsRed2 and NCI / ADR-Res-EGFP increased by 215-fold and 6-fold relative to day 1, respectively. In contrast, at the 5000-cell seeding density, OVCAR8- DsRed2 and NCVADR-RES-EGFP cell fluorescence changed by 48-fold and 1-fold. However, regardless of seeding density, the parental-to-subline growth ratio remains remarkably consistent throughout the study (Figure 3E).

[0112] A dose-response studies of NP-Tal in the spheroid coculture model was established to evaluate the role of talazoparib dose in spheroid evolution (Figure 4). On the lower dose range, the NCI / ADR-RES-EGFP cell line is spared throughout the study, while the parental OVCAR8-DsRed2 line succumbs to the treatment. In contrast, higher doses kill both cell lines to equivalent degrees. This demonstrates a trend towards acquired chemoresistance where sublethal treatment is applied, a phenomenon consistently observed in prior studies

[0024] , Next, the PIC -NP-Tal nanocomplex is tested in the 3D coculture model (Figure 5). PIC mixed with NP-Tal significantly outperformed the nanocomplex (PIC-NP-Tal) in spheroid killing at 20 and 50 J / cm2, though both treatments drove chemoresistance. Similarly, treatment with PIC alone and BPD+NP-Tal both drove chemoresistance. In contrast, NP-Tal and BPD as monotherapies were the only groups to kill both the parental and resistant subline to equivalent degrees across all light doses, thereby avoiding domination of the chemo-resistant subline.

[0113] Example 12. Conclusions

[0114] Photoimmunotherapy and PARP inhibition are clinically relevant cancer treatment modalities with synergistic potential. In this study, these modalities are combined to achieve a novel nanocomplex for codelivery of Cetuximab-BPD PICs and talazoparib. First, formulation parameters were optimized to establish a polymeric nanoparticle loaded with talazoparib with capabilities for click chemistry to attach PIC. The PIC-to-nanoparticle reaction ratio was next optimized, and the formulation was thoroughly characterized for photochemical and biological properties. In parallel, a 3D model of ovarian cancer with fluorescently labeled chemosensitive (OVCAR8-DsRed2) and chemo-resistant (NCI / ADR-RES-EGFP) subpopulations was developed and tracked up to 12 days. Treatment of spheroids with varying doses of NP- Tal revealed that lower doses induce selection pressures in favor of the chemo-resistant subline, whereas higher doses are similarly cytotoxic to both cell lines. Evaluation of PIC-NP-Tal in the 3D spheroid model revealed inferior therapeutic effects compared to co-treatment of PICand NP-Tal. Additionally, PIC, BPD+NP-Tal, PIC+NP-Tal, and PIC-NP-Tal all drove chemoresistance, whereas NP-Tal and BPD as monotherapies did not. Overall, these data provide new insights into combinational therapies in the context of 3D spheroids, indicating that conjugation of multiple therapeutic entities may not always outperform the unconjugated combination. Results from this study also indicate that while combinational therapies may enhance total cell killing compared to monotherapies, they may also drive chemoresistance, reinforcing the fundamental importance of preclinical models of multidrug resistance.

[0115] All publications mentioned herein are incorporated by reference to the extent they support the present invention.

[0116] REFERENCES1. Schreiber V, Dantzer F, Ame J-C, de Murcia G: Poly(ADP-ribose): novel functions for an old molecule. Nature Reviews Molecular Cell Biology 2006, 7(7): 517-528.2. Javle M, Curtin NJ: The role of PARP in DNA repair and its therapeutic exploitation. Br J Cancer 2011, 105(8): 1114-1122.3. Ray Chaudhuri A, Nussenzweig A: The multifaceted roles of PARP 1 in DNA repair and chromatin remodelling. Nat Rev Mol Cell Biol 2017, 18(10):610-621.4. Rose M, Burgess JT, O’Byrne K, Richard DJ, Bolderson E: PARP Inhibitors: Clinical Relevance, Mechanisms of Action and Tumor Resistance. Frontiers in Cell and Developmental Biology 2020, 8.5. Valabrega G, Scotto G, Tuninetti V, Pani A, Scaglione F: Differences in PARP Inhibitors for the Treatment of Ovarian Cancer: Mechanisms of Action, Pharmacology, Safety, and Efficacy. IntJMol Sci 2021, 22(8).6. Sandhu D, Antolin AA, Cox AR, Jones AM: Identification of different side effects between PARP inhibitors and their polypharmacological multi-target rationale. British Journal of Clinical Pharmacology 2022, 88(2)742-752.7. Boussios S, Abson C, Moschetta M, Rassy E, Karathanasi A, Bhat T, Ghumman F, SheriffM, Pavlidis N: Poly (ADP -Ribose) Polymerase Inhibitors: Talazoparib in Ovarian Cancer and Beyond. Drugs R D 2020, 20(2):55-73.8. Pommier Y, O’Connor MJ, de Bono J: Laying a trap to kill cancer cells: PARP inhibitors and their mechanisms of action. Science Translational Medicine 2016, 8(362):362ps317-362ps317.Mokhtari RB, Homayouni TS, Baluch N, Morgatskaya E, Kumar S, Das B, Yeger H: Combination therapy in combating cancer. Oncotarget 2017, 8(23). Sorrin AJ, Kemal Ruhi M, Ferlic NA, Karimnia V, Polacheck WJ, Celli JP, Huang H- C, Rizvi I: Photodynamic Therapy and the Biophysics of the Tumor Microenvironment. Photochemistry and Photobiology 2020, 96(2):232-259. Baglo Y, Sorrin AJ, Pu X, Liu C, Reader J, Roque DM, Huang H-C: Evolutionary dynamics of cancer multidrug resistance in response to olaparib and photodynamic therapy. Translational Oncology 2021, 14(11): 101198. Tanaka M, Sasaki M, Suzuki T, Nishie H, Kataoka H: Combination of talaporfm photodynamic therapy and Poly (ADP -Ribose) polymerase (PARP) inhibitor in gastric cancer. Biochem Biophys Res Commun 2021, 539: 1-7. Lei S, Ge F, Lin M, Wang X, Shen J, Yang Y, Deng J, Wang Z, Wang J, Li K: PARP inhibitors diminish DNA damage repair for the enhancement of tumor photodynamic therapy. Photodiagnosis and Photodynamic Therapy 2022, 40: 103058. Magalhaes JA, Arruda DC, Baptista MS, Tada DB: Co-Encapsulation of Methylene Blue and PARP-Inhibitor into Poly(Lactic-Co-Glycolic Acid) Nanoparticles for Enhanced PDT of Cancer. Nanomaterials 2021, 11(6): 1514. Rottenberg S, Jaspers JE, Kersbergen A, van der Burg E, Nygren AO, Zander SA, Derksen PW, de Bruin M, Zevenhoven J, Lau A et al. High sensitivity of BRCA1- deficient mammary tumors to the PARP inhibitor AZD2281 alone and in combination with platinum drugs. Proc Natl Acad Sci USA 2008, 105(44): 17079-17084. Oplustilova L, Wolanin K, Mistrik M, Korinkova G, Simkova D, Bouchal J, Lenobel R, Bartkova J, Lau A, O'Connor MJ et al. Evaluation of candidate biomarkers to predict cancer cell sensitivity or resistance to PARP-1 inhibitor treatment. Cell Cycle 2012, ll(20):3837-3850. Liang BJ, Pigula M, Baglo Y, Najafali D, Hasan T, Huang HC: Breaking the selectivity-uptake trade-off of photoimmunoconjugates with nanoliposomal irinotecan for synergistic multi-tier cancer targeting. J Nanobiotechnology 2020, 18(1): 1-14. Singh B, Yang S, Krishna A, Sridhar S: Nanoparticle Formulations of Poly (ADP- ribose) Polymerase Inhibitors for Cancer Therapy. Front Chem 2020, 8:594619. Sargazi S, Mukhtar M, Rahdar A, Barani M, Pandey S, Diez -Pascual AM: Active Targeted Nanoparticles for Delivery of Poly(ADP -ribose) Polymerase (PARP) Inhibitors: APreliminary Review. IntJMol Sci 2021, 22(19).20. Cai L, Xu X, Chen W: The Current State of the Art in PARP Inhibitor-Based Delivery Nanosystems. In: Pharmaceutics, vol. 14; 2022.21. Spring BQ, Abu- Yousif AO, Palanisami A, Rizvi I, Zheng X, Mai Z, Anbil S, Sears RB, Mensah LB, Goldschmidt R et ah. Selective treatment and monitoring of disseminated cancer micrometastases in vivo using dual-function, activatable immunoconjugates. Proceedings of the National Academy of Sciences 2014, lll(10):E933-E942.22. Huang H-C, Pigula M, Fang Y, Hasan T: Immobilization of Photo-Immunoconjugates on Nanoparticles Leads to Enhanced Light- Activated Biological Effects. Small 2018, 14(31):1-11.23. Gomes-da-Silva LC, Kepp O, Kroemer G: Regulatory approval of photoimmunotherapy: photodynamic therapy that induces immunogenic cell death. Oncoimmunology 2020, 9(1): 1841393.24. Wurz GT, DeGregorio MW: Activating adaptive cellular mechanisms of resistance following sublethal cytotoxic chemotherapy: Implications for diagnostic microdosing. International Journal of Cancer 2015, 136(7): 1485-1493.

[0117] A number of patents and publications are cited above in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Full citations for these references are provided below. Each of these references is incorporated herein by reference in its entirety into the present disclosure, to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference.

[0118]

Claims

CLAIMSWe claim:

1. A poly (ADP-ribose) polymerase (PARP) inhibitor nanoparticle composition, said composition comprising a PARP inhibitor and a polymer, wherein said composition is in the form of a nanoparticle particle.

2. The composition of claim 1, wherein said nanoparticle has a size of about 50 nm to about 500 nm.

3. The composition of any of the preceding claims, wherein said PARP inhibitor is talazoparib, olaparib, rucaparib, veliparib, and niraparib, or a combination thereof.

4. The composition of any of the preceding claims , wherein said polymer is poly-lactic acid (PLA), poly(lactide-co-glycolide) (PLGA), poly(lactide-co-glycolide)-block- poly(ethylene glycol) (PLGA-PEG), poly(lactide-co-glycolide)-block-poly(ethylene glycol)-carboxylic acid (PLGA-PEG-COOH), or poly(lactide-co-glycolide)-block- poly(ethylene glycol)-dibenzocyclooctyne-amine (PLGA-PEG-DBCO).

5. The composition of claim 4, wherein said polymer is poly(lactide-co-glycolide)-block- poly(ethylene glycol)-carboxylic acid (PLGA-PEG-COOH), or poly(lactide-co- glycolide)-block-poly(ethylene glycol)-dibenzocyclooctyne-amine (PLGA-PEG- DBCO).

6. The composition of any of the preceding claims , wherein said composition further comprises a photosensitizer.

7. The composition of claim 6, wherein said photosensitizer is coupled to the surface of said nanoparticle composition via an antibody conjugate (i.e., antibody-photosensitizer conjugate a / k / a photoimmunoconjugate).

8. The composition of claims 6-7, wherein said photosensitizer is a benzoporphyrin derivative (BPD) photosensitizer, a chlorin-based photosensitizer, a porphyrin-based photosensitizer, a bacteriochlorin-based photosensitizer, a phthalocyanine-based photosensitizer, a ruthenium-based photosensitizer, or a fluorescence imaging agent.

9. A method of preparing a PARP inhibitor nanoparticle composition, said method comprising: dissolving a PARP inhibitor and a polymer in a water-miscible solvent (e.g., acetone) to obtain a PARP inhibitor-polymer solution; and adding to surfactant-containing water to obtain a mixture and sonicating said mixture;removing said solvent (e.g., evaporation) to obtain said PARP inhibitor-polymer nanoparticle.

10. The method of claim 9, wherein said PARP inhibitor has a concentration of about 0 mg / mL to about 100 mg / mL.

11. The method of claim 9, wherein said polymer is chosen from PLA, PLGA, PLGA-PEG, PLGA-PEG-COOH and PLGA-PEG-DBCO, or a combination thereof.

12. The method of claim 11, wherein said polymer is a combination of PLA, PLGA, PLGA- PEG, PLGA-PEG-COOH and PLGA-PEG-DBCO.

13. The method of claim 12, wherein the mass of said PLGA-PEG-DBCO to PLGA-PEG- COOH ratio is from 0: 100 to 100:0.

14. The method of claim 9, wherein said water-miscible solvent is added to water in the amount of about 0.1 %v / v to about 50 %v / v.

15. The method of claim 14, wherein said water-miscible solvent is added to water in the amount of about 10% v / v.

16. The method of claim 9, wherein said water-miscible solvent is acetone, ethanol, acetonitrile, anisole, chloroform, dichloro-methane, dimethylformamide, dimethylsulfoxide, ethyl acetate, or dioxane.

17. The method of claim 9, wherein said water-miscible solvent is acetone.

18. The method of claim 9, wherein said surfactant containing water comprises about 0.01% surfactant to about 10% surfactant.

19. The method of claim 9, wherein said surfactant is pluronics.

20. The method of claim 19, wherein said pluronics is Pluronic F-68.

21. The method of any of the preceding claims, further comprising adding an antibodyphotosensitizer conjugate to a surface of said PARP inhibitor-polymer nanoparticle to obtain a conjugated nanoparticle.

22. The method of claim 21, wherein the molar ratio of said antibody -photosensitizer conjugate to said PARP inhibitor-polymer nanoparticle is about 1 : 1 to about 1000: 1.

23. The method of claim 21, wherein said antibody-photosensitizer conjugate and said PARP inhibitor-polymer nanoparticle are mixed overnight.

24. The method of claim 23, wherein said antibody-photosensitizer conjugate and said PARP inhibitor-polymer nanoparticle are mixed for about 0.1 hours to about 24 hours.

25. The method of claim 21, wherein said antibody-photosensitizer conjugate is added to said surface of said PARP-polymer nanoparticle using copper-free click chemistry.

26. The method of claim 21, wherein said conjugated nanoparticle is purified by size exclusion chromatography.

27. A method for the treating cancer, said method comprising administering a nanoparticle composition of any of the preceding claims and optionally photosensitizer to a subject.

28. A method for the treating cancer, said method comprising administering a nanoparticle composition of any of the preceding claims and photosensitizer of any of the preceding claims to a subject.

29. The method of claim 27, wherein said composition and said photosensitizer are coadministered separately.

30. The method of claim 27, wherein said photosensitizer is coupled to the surface of said nanoparticle composition via an antibody conjugate.

31. The method of any of the preceding claims, said method further comprising subjecting said subject to photodynamic therapy or photoimmunotherapy.

32. The method of claim 27, wherein said cancer is an epidermal growth factor receptor (EGFR) expressing cancer.

33. The method of claim 27, wherein said cancer is a programmed death-ligand 1 (PD-L1) expressing cancer.

34. The method of claim 27, wherein said cancer is selected from the group consisting of gynecologic cancer, pancreatic cancer, prostate cancer, brain cancer, liver cancer, gastrointestinal cancer, head and neck cancer, skin cancer, soft tissue sarcoma, eye cancer, lung cancer, kidney cancer, esophageal cancer, peritoneal carcinoma, colorectal cancer, stomach cancer, peritoneal carcinomatosi , ovarian cancer, oral cancer, mesothelioma, and breast cancer.

35. The method of any of the preceding claims, wherein said composition are administered via a route chosen from oral administration, intravenous, intra-arterial, intratumoral, intravesical, intramuscular, subcutaneous, and intraperitoneal injections.

36. The method of any of the preceding claims, wherein said photosensitizer is administered via a route chosen from oral administration, intravenous, intra-arterial, intratumoral, intravesical, intramuscular, subcutaneous, and intraperitoneal injections.

37. The method of claim 27, wherein said photosensitizer coupled to the surface of said nanoparticle composition via an antibody conjugate is administered via a route chosen from oral administration, intravenous, intra-arterial, intratumoral, intravesical, intramuscular, subcutaneous, and intraperitoneal injections.

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