Compositions and methods for targeted delivery of combinatorial therapeutics

Abstract

Provided are compositions and methods that include use of micro-RNAs and mimics of the micro-RNAs for treating cancer. Specific ratios of micro-RNAs that have enhanced anti-cancer properties for treating prostate, bladder, and head and neck cancer are provided. The micro-RNAs are conjugated to nanoparticles configured so that exposure to light releases the micro-RNAs from the nanoparticles, thereby allowing the microRNAs to elicit anti-cancer effects.

Description

[0001] COMPOSITIONS AND METHODS FOR TARGETED DELIVERY OF COMBINATORIAL THERAPEUTICS CROSS REFERENCE TO RELATED APPLICATION

[0002] This application claims priority to U.S. provisional patent application no. 63 / 710,319, filed October 22, 2024, the entire disclosure of which is incorporated herein by reference.

[0003] SEQUENCE LISTING

[0004] The instant application contains a Sequence Listing which has been submitted in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on October 21, 2025, is named “074339_00334_ST26.xml”, and is 20,496 bytes in size

[0005] BACKGROUND

[0006] There is an ongoing and unmet need for improved compositions and methods for treating and inhibiting development of cancer. For instance, bladder cancer is currently the 10th most prevalent cancer globally and over the last 30 years has maintained its characterization as having a high propensity for recurrence and progression. In 2020, there were approximately 573,000 new cases of bladder cancer and 213,000 fatalities caused by this cancer type alone. As population, aging, social and economic growth, smoking rates, and environmental pollution are expected to increase, the incidence of bladder cancer in many countries around the world are expected to rise significantly in the coming decades, potentially bringing an even heavier disease burden. Likewise, prostate cancer is currently the most prevalent cancer among men in 121 of 185 total countries and has maintained its mortality rate over this past decade. In 2020, there were approximately 1,400,000 new cases of prostate cancer and 375,000 fatalities caused by this cancer type alone. As population, aging, obesity, low vitamin intake, and changes in the hormonal profile are expected to increase, the incidence of prostate cancer in many countries around the world are expected to maintain or rise slightly in the coming decades, potentially bringing a heavier disease burden. Similarly, for head and neck cancer, in 2020, it was the seventh most common cancer globally, making up about 4.5% of all cancer diagnoses worldwide. Thus, there is an ongoing unmet needs for new approaches to treating these cancers. The present disclosure is pertinent to this need.

[0007] BRIEF SUMMARY

[0008] The disclosure provides compositions and methods for treating cancer. The disclosure comprises microRNAs (miRs) at described ratios conjugated to nanoparticles. A ratio for any described combination of two miRs can be 50:50, 10:90, and 90:10, inclusive, and including all numbers and ranges of numbers there between for each described combination. In examples, the described miR combinations are provided in a molar ratio of 50:50, 40:60, 30:70, 20:80, 10:90, 90:10, 80:20, 70:30, or 60:40.

[0009] The disclosure provides in one example compositions and methods for treating head and neck cancer using miR-148b-3p and miR-34a-5p or mimics thereof at a 10(miR-148b-3p):90(miR-34a-5p) molar ratio. Both the miR-148b-3p and the miR-34a-5p or mimics thereof are conjugated to each particle in a plurality of particles in the described molar ratio. Thus, each particle in the plurality of particles has both microRNAs or mimics thereof conjugated to it. The plurality of particles is administered to an individual who has been diagnosed with head and neck cancer. The miRNAs are released from the nanoparticles by stimulation with a suitable light source.

[0010] In another example, the disclosure provides compositions and methods for treating bladder cancer using miR-3619-5p and miR-26b-5p or mimics thereof at 50:50 ratio. Both the miR-3619-5p and miR-26b-5p or mimics thereof are conjugated to each particle in a plurality of particles in the described molar ratio. The plurality of particles is administered to an individual who has been diagnosed with bladder cancer. The miRNAs are released from the nanoparticles by stimulation with a suitable light source.

[0011] In another example, the disclosure provides compositions and methods for treating prostate cancer using miR-34a-5p and miR-222-3p or mimics thereof at a 90(miR-34a-5p):10(miR-222-3p) ratio. Both the miR-34a-5p and miR-222-3p or mimics thereof are conjugated to each particle in a plurality of particles in the described molar ratio. The plurality of particles is administered to an individual who has been diagnosed with prostate cancer. The miRNAs are released from the nanoparticles by stimulation with a suitable light source.

[0012] The disclosure demonstrates that using the described molar ratios of the microRNAs and mimics thereof is improved relative to using the microRNAs or mimics alone, or together but in different ratios. The disclosure also demonstrates that the described particles only exert their anti-cancer effects when exposed to a suitable wavelength of light. As such, the disclosure provides approaches to treating cancers that are on the epidermis or within 3 centimeters of the epidermis, or are accessible using intra-body devices such as endoscopes or catheters adapted to provide a suitable light source that liberates the miRNAs from the nanoparticles, which in turn facilitates anti-cancer activity of the miRNAs. Pluralities of nanoparticles that are conjugated to the described miRNAs in the described ratios on each nanoparticle are provided.

[0013] BRIEF DESCRIPTION OF THE FIGURES

[0014] Where used in the Figures and the description of this disclosure, “CSS” means core-shell-shell (CSS) nanoparticles.

[0015] FIG. 1. Surface Plasmonic Resonance and Diels-Alder showing effect of wavelengths of light (panel A) and effect on a described miRNA conjugated nanoparticle (NP) (panel B).

[0016] FIG. 2. Nanoparticle (NP) synthesis and miRNA overview showing synthesis scheme (panel A) and conjugation of control and representative mir-34a-5p and mir-148b-3p onto nanoparticles (panel B). Both miRNA are conjugated to single nanoparticles in a described ratio.

[0017] FIG. 3. Nanoparticle sizes and intensity (panel A) and % of sizes (panel B) for representative nanoparticles.

[0018] FIG. 4. Graph showing NP responsiveness to different wavelengths.

[0019] FIG. 5. Graph summarizing miRNA release in water and serum at different temperatures.

[0020] FIG. 6. Fluorescence images showing effects of miR-148b-3p, miR-34a-5p, and combinations on FaDu cells at different time points using Lipofectamine delivery.

[0021] FIG. 7. Fluorescence images demonstrating how varying ratios of miR-34a and miR-148b impacts suppression in FaDu cells at 100 nM.

[0022] FIG. 8. Graphs summarizing data demonstrating that lipofectamine delivery of combinatorial miRNA can induce apoptosis in FaDu cells.

[0023] FIG. 9. Fluorescence images showing lipofectamine delivery of combinatorial miRNA can induce apoptosis in FaDu cells at 100 nM.

[0024] FIG. 10. Graphs summarizing data showing lipofectamine delivery of combinatorial miRNA can induce apoptosis in FaDu cells at 100 nM where panel A shows results from a plate reader of L / D at 100 nm and panel B shows results using PicoGreen of 100 nM.

[0025] FIG. 11. Graphs summarizing results showing that 90-120 seconds of irradiation releases most miRNA from 1 mL of CSS NPs.

[0026] FIG. 12. Fluorescence images demonstrating CSS delivery of combinatorial miRNA without irradiation does not cause significant death.

[0027] FIG. 13. Fluorescence images demonstrating CSS delivery of combinatorial miRNA with irradiation induces apoptosis in FaDu cells at 50 nM. FIG. 14. Quantitative analysis of CSS delivery shows significant knockdown in survival when exposed to near-infrared (nIR) irradiation (panel B with light) compared to panel A (no light).

[0028] FIG. 15. Graphs summarizing results showing gene expression of apoptosis targets at 3 hours and 12 hours using miRNA as indicated.

[0029] FIG. 16. Cartoon illustration design of combinatorial murine xenograft animal model treated with described NPs and the effects of nIR irradiation on the CSS-miRNA.

[0030] FIG. 17. Cartoon flowchart of design and treatment of murine xenograft model.

[0031] FIG. 18. Graphs showing no significant change in murine model weight after short term (panel A) or long-term (panel B) treatment with the indicated miRNAs.

[0032] FIG. 19. Graphs showing results on tumor size with no light (panel A) or with application of light (panel B).

[0033] FIG. 20. Graphs showing tumor size (panel A) and Survival Percentage (panel B) after treatment with the indicated miRNAs. Significance between groups is summarized in the table of panel C.

[0034] FIG. 21. Graphs showing the effect on mouse weight (panel A) and tumor size (panel B) using the indicated miRNAs.

[0035] FIG. 22. Fluorescence images demonstrating the effects of combinatorial delivery of miR-26b-5p and miR-3619-5p to Bladder Cancer (5637 cell line) using lipofectamine.

[0036] FIG. 23. Graphs showing the indicated miRNAs in the indicated ratios in 5637 cells using lipofectamine as the delivery agent. Results show surviving cell percentages over time.

[0037] FIG. 24. Graphs showing the indicated miRNAs in the indicated ratios in 5637 cells using lipofectamine as the delivery agent. Results show surviving cell percentages over time.

[0038] FIG. 25. Fluorescence images demonstrating CSS delivery of combinatorial miRNA with and without treatment for the indicated miRNAs.

[0039] FIG. 26. Graphs showing results obtained using the indicated miRNAs and CSS without nIR irradiation.

[0040] FIG. 27. Graphs showing results obtained using the indicated miRNAs and CSS with nIR irradiation.

[0041] FIG. 28. Graphs showing results obtained using the indicated miRNAs using lipofectamine as the delivery agent, using the indicated miRNAs in prostate cancer cells.

[0042] FIG. 29. Graphs showing results obtained using the indicated miRNAs using lipofectamine as the delivery agent, using the indicated miRNAs in prostate cancer cells. FIG. 30. Graphs showing results obtained using the indicated miRNAs using CSS NPs without irradiation.

[0043] FIG. 31. Graphs showing results obtained using the indicated miRNAs using CSS NPs with irradiation.

[0044] FIG. 32. Cartoon flowchart illustrating steps of in vivo animal testing.

[0045] FIG. 33. Graphs showing results on animal weight using CSS and the indicated miRNAs.

[0046] FIG. 34. Graphs showing results on tumor volume without irradiation (left) and with irradiation (right) using CSS and the indicated miRNAs.

[0047] FIG. 35. Graphs showing results from treatment using the indicated miRNAs on tumor size (left) and survival (right).

[0048] FIG. 36. Graphs showing results from treatment using the indicated miRNAs on mouse weight (left) and tumor size (right).

[0049] FIG. 37. Sequences of miRNA mimics used to produce data described in previous figures, “m” signifies 2’ O-methyl modification (2’-M0E).

[0050] DETAILED DESCRIPTION

[0051] 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 belongs.

[0052] Every numerical range given throughout this specification includes its upper and lower values, as well as every narrower numerical range that falls within it, as if such narrower numerical ranges were all expressly written herein.

[0053] As used in the specification and the appended claims, the singular forms “a” "and” and “the" include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another example includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about” it will be understood that the particular value forms another example. The term “about” in relation to a numerical value encompasses variations of + / - 10%, + / - 5%, or + / - 1%.

[0054] This disclosure is related in part to U.S. Patent No. 11,559,494 B2, and PCT publication No. WO 2023 / 114466 Al, the entire disclosures of each of which are incorporated herein by reference. In examples, the disclosure relates to use of combinations of therapeutic species, such as combinations of different therapeutic polynucleotides and derivatives thereof wherein different therapeutic polynucleotides and / or derivatives thereof are joined via a linker to a nanoparticle, such as a metal nanoparticle. Pluralities of nanoparticles that may be joined to a single therapeutic polynucleotide or derivatives thereof, and nanoparticles that are joined to more than one type of therapeutic polynucleotide or derivative thereof, are included within the disclosure. In examples, the disclosure employs Light Inducible Gene Regulation (LIGeR) vehicles.

[0055] The disclosure includes use of nanoparticles conjugated to a therapeutic species as described in U.S. Patent No. 11,559,494, and WO2023114466, and as described herein and by way of the accompanying figures. In examples, the described combinatorial miRNA approach results in increased apoptosis, decreased proliferation, decreased adhesion, or a combination thereof, for cancer cells. In examples, a change in a described effect using a combinatorial approach is greater than the same effect but obtained using a single therapeutic species or a different ratio of two therapeutic species.

[0056] In examples, by combining miRNA mimics targeting orthogonal genetic pathways in cancer cells, the present disclosure allows for a reduction of the total dose of therapeutic species and kills cancer cells that do not respond to a single miRNA mimics alone, or to combinations of miRNAs but in different molar ratios compared to the molar ratios exemplified as having improved therapeutic efficacy as described herein. In examples, the described approach provides for precision spatiotemporal control of nucleic acid therapeutics and by light induced release of the described nucleic acid therapeutics.

[0057] In examples, the disclosure demonstrates the following: synthesizing and characterizing the LIGeR delivery vehicles; the LIGeR vehicle can transfect cancer cells and selectively drive apoptosis; and miRNAs as will be apparent from the figures are identified as non-apoptotic in solo treatment and in certain ratios as apoptotic in combinatorial treatment of head and neck cancer cell lines, compared to the preferred ratios as further described herein.

[0058] The disclosure includes using the described miroRNAs or mimics thereof. The term “microRNA” can be used interchangeably with “miR,” or “miRNA” to refer to, for example, an unprocessed or processed RNA transcript from a naturally occurring or engineered miRNA gene. The unprocessed miRNA gene transcript is also called a “miRNA precursor,” and typically comprises an RNA transcript of about 70-100 nucleotides in length. The miRNA precursor can be processed by digestion with an RNAse (for example, Dicer, Argonaut, or RNAse III) into an active 19-25 nucleotide RNA molecule. This active 19-25 nucleotide RNA molecule is also called the “processed” miRNAgene transcript or “mature” miRNA. Any of these forms of microRNA can be adapted for use in embodiments of this disclosure.

[0059] In examples, the microRNA may be provided as a synthetic agent, such as a microRNA mimic. In examples, a described mimic comprises a modified ribosugar modification, a modified ribonucleotide, a modified inter-nucleoside linkage, or a combination thereof. In examples, a modification comprises a 2 '-deoxy-2 '-fluoro (2'-F) ribosugar modification, a 2'-O-methyl (2'-0Me) ribosugar modification, a 2'-a GalNAc clicked cytidine, or a phosphorothioate linkage. The disclosure also includes use of modified ribonucleotides or deoxyribonucleotide, and thus include RNA / DNA hybrids. In non-limiting examples, modified ribonucleotides may comprise methylations and / or substitutions of the 2' position of the ribose moiety with an — O— alkyl group containing 1-6 saturated or unsaturated carbon atoms, or with an — O-aryl group having 3-6 carbon atoms, wherein such alkyl or aryl group may be unsubstituted or may be substituted, e.g., with halo, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl, carbalkoxyl, or amino groups; or with a hydroxy, an amino or a halo group. In examples modified nucleotides comprise methyl-cytidine and / or pseudo-uridine. The nucleotides may be linked by phosphodiester linkages or by a synthetic linkage, i.e., a linkage other than a phosphodiester linkage.

[0060] Examples of inter-nucleoside linkages in the polynucleotide agents that can be used in the disclosure include, but are not limited to, phosphodiester, alkylphosphonate, phosphorothioate, phosphorodithioate, phosphate ester, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, morpholino, phosphate triester, acetamidate, carboxymethyl ester, or combinations thereof. In an example, LIGeR-miRNA, synthesized with a 2-furanmethanethiol dienophile and maleimide diene is cytocompatible and can deliver functional nucleic acids intracellularly.

[0061] In examples, the described miRNA molar ratios are used with the miRNA mimics depicted in FIG. 37. The disclosure includes all ratios of the described miRNAs ratios conjugated to nanoparticles. A ratio for any described combination of two miRs can be 50:50, 10:90, and 90:10, inclusive, and including an numbers and ranges of numbers there between for each described combination. In examples, the described miR combinations are provided in a molar ratio of 50:50, 40:60, 30:70, 20:80, 10:90, 90: 10, 80:20, 70:30, or 60:40.

[0062] In one example, the disclosure provides compositions and methods for treating head and neck cancer using miR-148b-3p and miR-34a-5p or mimics thereof at a 10:90 molar ratio. Both the 148b-3p and the miR-34a-5p or the mimics thereof are conjugated to each particle in a plurality of particles in the described molar ratio. In an example, the hsa-miR-148b-3p mimic comprises or consists of SEQ ID NO:3. In an example, the miR-34a-5p mimic comprises or consists of SEQ ID NO:2. Each particle in the plurality of particles has both microRNA mimics conjugated to it. The plurality of particles is administered to an individual who has been diagnosed with head and neck cancer. Upon light stimulation, the miRNA mimics are released from the nanoparticles and exert their anti-cancer function.

[0063] In another example, the disclosure provides compositions and methods for treating bladder cancer using miR-3619-5p and miR-26b-5p or mimics thereof at 50:50 ratio. Both the miR-3619-5p and miR-26b-5p or mimics thereof are conjugated to each particle in a plurality of particles in the described molar ratio. In an example, the miR-3619-5p mimic comprises or consists of SEQ ID NO:5. In an example, the miR-26b-5p mimic comprises or consists of the sequence of SEQ ID NO:6. The plurality of particles is administered to an individual who has been diagnosed with bladder cancer. Upon light stimulation, the miRNA mimics are released from the nanoparticles and exert their anti-cancer function.

[0064] In another example, the disclosure provides compositions and methods for treating prostate cancer using miR-34a-5p and miR-222-3p or mimics thereof at a 90: 10 ratio. Both the miR-34a-5p and miR-222-3p or mimics thereof are conjugated to each particle in a plurality of particles in the described molar ratio. In an example, the miR-34a-5p mimic comprises or consists of the sequence of SEQ ID NO:2. In an example, the mir-222-3p mimic comprises or consists of the sequence of SEQ ID NO:4. The plurality of particles is administered to an individual who has been diagnosed with prostate cancer. Upon light stimulation, the miRNAs are released from the nanoparticles and exert their anti-cancer function.

[0065] The disclosure demonstrates that using the described molar ratios of the microRNAs or mimics thereof is improved relative to different miRNA ratios for the same described miRNA pairs. The disclosure also demonstrates that the described particles only exert their anti-cancer effects when exposed to a suitable wavelength of light. As such, the disclosure provides approaches to treating cancers that are on the epidermis or within 3 centimeters of the epidermis, or are accessible using intra-body devices such as endoscopes or catheters adapted to provide a suitable light source.

[0066] In examples, a composition comprising a miRNA or mimic thereof conjugated to a particle is administered to an individual in a therapeutically effective amount. The term “therapeutically effective amount” as used herein refers to an amount of a described agent sufficient to achieve, in a single or multiple doses, the intended purpose of treatment. The amount desired or required may vary depending on the particular microRNA-conjugated particles, the mode of administration, patient specifics and the like. Appropriate effective amounts can be determined by one of ordinary skill in the art informed by the instant disclosure using routine experimentation. For example, a therapeutically effective amount, e.g., a dose, can be estimated initially either in cell culture assays or in animal models. An animal model can also be used to determine a suitable concentration range, and route of administration. A precise dosage can be selected by in view of the patient to be treated.

[0067] Dosage and administration can be adjusted to provide sufficient levels of components to achieve a desired effect. Factors which may be taken into account include the type of condition, the age, weight and gender of the patient, desired duration of treatment, method of administration, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance / response to therapy. In certain examples, a therapeutically effective amount is an amount that reduces one or more signs or symptoms of a disease, and / or reduces the severity of the disease. A therapeutically effective amount may also inhibit or prevent the onset of a disease, or a disease relapse. In examples, a therapeutically effective amount is an amount that inhibits growth of a tumor. In examples, an therapeutically effective amount inhibits metastasis from a primary tumor. In examples, a therapeutically effective amount extends the lifespan of the individual, relative to the average lifespan of individuals who did not receive a described treatment.

[0068] In examples, the particles of this disclosure may have only the described microRNAs or mimics thereof conjugated to them, and only in the described ratios.

[0069] Certain aspects of the disclosure are illustrated by the figures. As will be apparent from the figures, in examples, two different microRNAs or mimics thereof may be conjugated to each particle in a plurality of particles, and in the described molar ratios. In examples, the microRNAs or mimics thereof are conjugated to a metal nanoparticle and wherein a linker joins the metal nanoparticle to the microRNAs or mimics thereof.

[0070] The linker is not particularly limited and can be any linker that is not inconsistent with the objectives of this disclosure. In examples, a linker comprises a Diels- Alder cyclo-addition product. A Diels- Alder reaction is a conjugate addition reaction of a conjugated diene with a dienophile. In an example, the linker comprises a Diels-Alder cyclo-addition reaction product. In an example, the Diels-Alder cyclo-addition reaction product has a retro Diels-Alder reaction activation temperature between 30° C. and 170° C. In an example, the metal nanoparticle has a plasmon resonance frequency in a visible region or in an infrared region of the electromagnetic spectrum. In an example, initiating the retro Diels-Alder reaction comprises heating the metal nanoparticle to the retro Diels- Alder reaction activation temperature of the composition. In an example, heating the metal nanoparticle to the activation temperature comprises exposing the metal nanoparticle to electromagnetic radiation comprising a frequency corresponding to the plasmon resonance frequency of the metal nanoparticle.

[0071] The metal nanoparticles can be formed from any suitable metal, such as any metal that can be heated by an external stimulus or signal (such as light). For example, in some examples, a nanoparticle described herein is formed from silver, gold, platinum, or a mixture or alloy thereof. In some examples, the metal nanoparticles are “plasmonic” metal nanoparticles, particularly metal nanoparticles having a plasmon resonant frequency at a wavelength of light suitable for use in a biological environment, such as visible light, infrared (IR) light, or near infrared (NIR). For example, the visible light in some instances is light corresponding to wavelengths from 300 nm to 700 nm; 390 nm to 700 nm; 390 nm to 600 nm; 390 nm to 500 nm; 390 nm to 450 nm; 450 nm to 700 nm; 500 nm to 650 nm; 550 nm to 600 nm; 500 nm to 700 nm; 550 nm to 650 nm; or 600 nm to 700 nm. The infrared light in some instances is light corresponding to wavelengths from 700 nm to 1 mm; 800 nm to 900 nm; 900 nm to 800 nm; 1000 pm to 700 nm; 900 pm to 800 pm; 700 nm to 900 pm; 700 nm to 800 pm; 700 nm to 700 pm; 700 nm to 600 pm; 700 nm to 500 pm; 700 nm to 400 pm; 700 nm to 300 pm; 700 nm to 200 pm; 700 nm to 100 pm; 700 nm to 1000 nm; 700 nm to 900 nm; or 700 nm to 800 nm. The near infrared light in some instances is light corresponding to wavelengths from 700 nm to 1 pm (1000 nm); 750 nm to 950 nm; 800 nm to 900 nm; 750 nm to 1 mm; 800 nm to 1 mm; 850 nm to 1 mm; 900 nm to 1 mm; 950 nm to 1 mm; 700 nm to 950 nm; 700 nm to 900 nm; 700 nm to 850 nm; 700 nm to 800 nm; or 700 nm to 750 nm.

[0072] In examples, a nanoparticle of a composition described herein has a size or diameter of 1-500 nm, 1-300 nm, 1-200 nm, 1-100 nm, 1-50 nm, 1-30 nm, 1-10 nm, 10-500 nm, 10-300 nm, 10-200 nm, 10-100 nm, 10-50 nm, 20-300 nm, 20-100 nm, 50-500 nm, 50-200 nm, or 50-100 nm in two dimensions or three dimensions. A population of nanoparticles of a composition described herein can also have an average size or diameter listed above.

[0073] In examples, a nanoparticle of a composition described herein can also have any. For example, in some cases, a nanoparticle described herein has a spherical or rod shape. Further, a nanoparticle can have a regular shape or an irregular shape.

[0074] This disclosure includes use of photo-thermal and / or magneto-thermal properties of nanoparticles described herein, such as metal and / or magnetic nanoparticles. As understood by one of ordinary skill in the art, these properties refer to a nanoparticle's ability to convert light (photo) energy or magnetic (magneto) energy to thermal energy, which heats the nanoparticle. Thermal energy from the nanoparticle can then be transferred to the linker, resulting in severing of the linker and decoupling of the therapeutic species from the nanoparticle.

[0075] One example of the conversion of light energy to thermal energy occurs in plasmonic metallic nanoparticles. Plasmonic metallic nanoparticles resonate (e.g., form resonant plasmons) at discrete photonic wavelengths of applied light. These resonant plasmons can decay into photons, which heat the particle in direct proportion to the photo capture cross section and the quantum efficiency of the plasmon-to-phonon conversion. Thus, heating of the plasmonic nanoparticles occurs primarily when a wavelength of the applied light matches the unique resonant frequency of the nanoparticles and forms a resonant plasmon.

[0076] Amain determinate of resonant frequency in these plasmonic metal nanoparticles is composition. For example, silver nanoparticles have a resonant frequency with a local maximum at about 420 mu, whereas gold nanoparticles resonate at about 535 nm, and platinum nanoparticles resonate at about 215 nm .

[0077] Another determinant of resonance frequency is morphology. As an example, nanorods have multiple resonant frequencies correlating to transverse and longitudinal modes. The longitudinal modes provide red shifted resonance frequencies in the near infrared (NIR) region of the spectrum. The transverse modes have much shorter wavelengths, typically found in the optical or visible region of the electromagnetic spectrum. Due to the discrete resonant frequencies of the plasmonic nanoparticles described herein, heating of the nanoparticles, and, as a result, severing of the linker and decoupling of the therapeutic species from the nanoparticle, can be controlled. In general, for heating of plasmonic nanoparticles to occur to a degree sufficient to sever a linker in a manner described herein, the frequency of applied light matches or substantially matches a resonance frequency of the plasmonic nanoparticle.

[0078] In examples, decoupling of the therapeutic species from the nanoparticle can be spatiotemporally controlled by using different color (frequency and wavelength) light for stimulation of the plurality of nanoparticles at different times or in different locations (e.g., different locations within an individual). The different light frequencies can each match or substantially match at least one of the resonance frequencies of the different plasmonic nanoparticles. In an example, the disclosure provides a plurality of nanoparticles wherein each nanoparticle in the plurality of nanoparticles comprises an miR-34a-5p or mimic thereof and an miR-222-3p or mimic thereof conjugated to each nanoparticle in the plurality of nanoparticles in a 90:10 molar ratio, respectively.

[0079] In an example, the disclosure provides a plurality of nanoparticles wherein each nanoparticle in the plurality of nanoparticles comprises miR-148b-3p or a mimic thereof and an miR-34a-5p or a mimic thereof conjugated to each nanoparticle the plurality of nanoparticles at a 10:90 molar ratio, respectively.

[0080] In an example, the disclosure provides a plurality of nanoparticles wherein each nanoparticle in the plurality of nanoparticles comprises miR-3619-5p or a mimic thereof and miR-26b-5p or a mimic thereof in a 50:50 molar ratio conjugated to each nanoparticle the plurality of nanoparticles at a 10:90 molar ratio, respectively.

[0081] In examples, the described nanoparticles are conjugated to miR mimics having the sequence identifiers as described herein, and in the described ratios.

[0082] The Examples of this disclosure are provided on the Figures. Data obtained and summarized by the figures were produced using the following materials and methods. The materials and methods are illustrated for the miR-34a-5p and miR-148b-3p for Head and Neck Cancer treatment. The same approaches were used for the miR-3619-5p and miR-26b-5p at 50:50 ratio for bladder cancer and for the miR-34a-5p and 10% miR-222-3p at a 90:10 ratio for prostate cancer. The figures show also show data from controls (such as by using a control miRNA of SEQ ID NO:1, or miRNAs alone, or miRNAs in different combinations of ratios, only some of which have desirable anti-cancer cell effects.

[0083] Materials

[0084] Materials for the Diels-Alder linker included 6-Maleimidohexanoic acid (MilliporeSigma 755842), methanol (Millipore Sigma 322415), and 2-furanmethanethiol (Millipore Sigma F20408). Materials for the core-shell-shell nanoparticles include gold (III) chloride (MilliporeSigma 334049), sodium citrate (MilliporeSigma S4641), ascorbic acid (MilliporeSigma A8960), silver nitrate (MilliporeSigma 209139), sodium hydroxide (Millipore Sigma S5881), hydroquinone (MilliporeSigma H17902), and RNase free water (Dot Scientific DS248700). Materials for the nanoparticle functionalization included N-hydroxysuccinimide (NHS) (MilliporeSigma 130672), 1,2-di chloroethane (also known as ethylene dichloride) and l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (ThermoFisher 22980), isopropanol (MilliporeSigma 19030), and miRNA mimics (custom Integrated DNA Technologies or IDT). Additional materials for nanoparticle characterization include copper 200 mesh grids with lacey carbon film (VWR 100491-184). Materials for cell culture included DMEM (ThermoFisher 11965118), fetal bovine serum (Fisher Scientific MT35015CV), penicillin / streptomycin (VWR 45000-653), trypsin / EDTA (VWR, 45000-664) and FaDu cells (ATCC HTB-43). Materials for transfection included Lipofectamine RNA iMAX (ThermoFisher 13778030), OPTI-MEM (ThermoFisher 11058021), and LIVE / DEAD Viability / Cytotoxicity kit (ThermoFisher L3224). Additional materials for visualizing uptake with confocal microscopy included CELLview glass bottom cell culture dishes (VWR 89125-442), phosphate buffered saline without calcium or magnesium (VWR 45000-434), and DAPI (ThermoFisher D1306). Additional materials used for testing cell viability included the Quant IT PicoGreen dsDNA assay kit (ThermoFisher Pl 1496) and proteinase K (MilliporeSigma 70663-4) for use with the PicoGreen kit. For gene expression analysis, additional materials included Trizol Reagent (ThermoFisher 15596026), chloroform (MilliporeSigma C2432-1L), spin column tubes from the Purelink RNAMini Kit (ThermoFisher 12183018A), Verso cDNA synthesis kit (ThermoFisher AB 1453 A), primers for RT-qPCR (custom order from IDT), and PowerUp Sybr Green Master Mix (ThermoFisher A25741). Additional materials for animal studies included Matrigel (Corning CB40234), ImL leur-lok syringes (Fisher Scientific 14-817-108), 27-gauge needles (VWRBD305109), 26-gauge needles (VWR BD305110), and 21-gauge needles (VWRBD-305165). Homozygous Foxnl<nu> mice were purchased through the Penn State Animal Resource Program (Jackson Laboratories 007850). Isoflurane and artificial tear eye ointment for animal studies were purchased from the Penn State Animal resource program. Additional materials for the processing and histological analysis of tissue samples include 10% neutral buffered formalin (VWR 77507-022), 100 proof ethanol through Penn State specific catalogue (VWR TX89125174PSU), xylene substitute (VWR 77507-036), paraffin wax (VWR 470045-564), histology cassettes (VWR 18000-130), histology cassette foam pads (VWR 60872-492), scintillation vials (Fisher Scientific 0334025P), microtome blades (Fisher Scientific 3053835), cover glass (VWR 48393-059), and microscope slides (VWR 89033-053). Hematoxylin, Eosin, staining buffers, and xylene substitute mounting solution were provided by the Penn State Microscopy Core Facility.

[0085] Equipment and software used for experiments includes a SpectraMax iD3 microplate reader from Molecular Devices (San Jose, CA, USA), precision compact oven from ThermoFisher Scientific (Waltham, MA, USA), Olympus 1X73 inverted fluorescent microscope (Shinjuku City, Tokyo, Japan), ThorLabs T-cube LED Driver (Newton, NJ, USA), ThorLabs 850nm 900mW mounted LED (Newton, NJ, USA), Zeiss LSM 880 Airy scan Fast with PicoQuant FLIM (Oberkochen, Germany), QIAGEN Ingenuity Pathway Analysis (Redwood City, CA, USA), TargetScan v8.0 (Cambridge, Massachusetts, USA), Nanodrop One C Microvolume UV-Vis Spectrophotometer from ThermoFisher Scientific (Waltham, MA, USA), QuantStudio 3 Thermocycler from ThermoFisher Scientific (Waltham, MA, USA), PrimerBank an online database developed at Harvard (Cambridge, MA, USA), Design and Analysis 2 software from ThermoFisher Scientific (Waltham, MA, USA), Leica EG1150 (Wetzlar, Germany), Shandon finesse paraffin microtome from Thermo Scientific (Waltham, MA, USA), Leica ST5010 (Wetzlar, Germany), FEI Talos F200X from Thermo Scientific (Waltham, MA, USA), Malvern Zetasizer Nano ZS instrument (Malvern, Worcestershire, United Kingdom), Agilent Cary 5000 UV-Vis spectrophotometer (Santa Clara, CA, USA), DepMap Protal by the Broad Institute (Cambridge, Massachusetts, USA), and GraphPad Prism (Boston, MA, USA). Equipment for animal studies such as the isoflurane vaporizer and heated pads were supplied by the Penn State Animal Resource Program in the vivarium facilities.

[0086] Methods

[0087] Diels- Alder Linker Synthesis

[0088] The furan-based Diels- Alder (FDA) linker was prepared 1 week prior to its use for nanoparticle functionalization. 1.6g of 6-Maleimidohexanoic acid was measured and added to a scintillation vial. 15mL of methanol was added to the vial. The solution was vortexed and sonicated as needed to dissolve the 6-Maleimidohexanoic acid. 380uL of 2-furanmethanethiol was added to the glass vial and vortexed until evenly mixed. The vial was wrapped with parafilm and aluminum foil. The vial was placed on the rocker at room temperature for 7 days, then used for downstream applications.

[0089] CSS Nanoparticle Synthesis

[0090] The reagents were weighed and suspended in RNase free water to produce desired stocks. Stocks were made fresh at least monthly. Gold chloride was suspended at 29mM. Sodium citrate was suspended at 34mM. Ascorbic acid was suspended at lOOmM. Silver nitrate was suspended at lOOmM. Sodium hydroxide was suspended at lOOmM.

[0091] Hydroquinone was suspended at 0.03M. The stocks were vortexed briefly and sonicated for 30 minutes at room temperature before use. First, the gold seeds were produced. In a round bottom flask with a magnetic stir bar, 28.8 mL RNase free water, 300uL of 29nM gold chloride, and 900uL of 34mM sodium citrate were combined. The round bottom flask was placed into an oil bath and attached to a reflux condenser. The solution was stirred vigorously at boiling temperatures until the it turned bright red. This was within 2.5 minutes of initial color change. The round bottom flask was removed from the reflux condenser and oil bath. Then, the round bottom flask was capped and allowed to cool to room temperature. Once cool, the size of the gold seeds was measured with DLS to ensure they were between 10-25 nm in diameter. The gold seeds were stored at 4°C or used immediately for downstream synthesis.

[0092] Second, the silver shell was added to the gold core. A magnetic stir bar and lOmL RNase free water were added to a 50mL conical centrifuge tube. The tube was placed on a stir plate and stirred vigorously to create a vortex in the solution. 200uL of the gold seeds were added and allowed to stir for 1 minute. 60uL of the lOOmM ascorbic acid stock was added and allowed to stir for 1 minute. 15uL of the lOOmM silver nitrate solution was added and allowed to stir for approximately 2 minutes until the color of the solution stopped changing. 75uL of lOOmM sodium hydroxide was added and allowed to stir for 2 minutes. The magnetic stir bar was removed from the tube, the tube was capped, and the core-shell particles were centrifuged at 4000rpm for 20 minutes. The supernatant was removed without disturbing the pellet of particles and resuspended in lOmL of RNase free water. If necessary, the tube was sonicated briefly to break up the particle pellet. The core-shell particles were used immediately for the synthesis of core-shell-shell particles, not stored at an intermediate phase.

[0093] Third, the outer gold shell was added to the core-shell particles. A magnetic stir bar was added to the tube, and the tube was placed on the stir plate with vigorous stirring to create a vortex in the solution. IOOUL of 29mM gold chloride was added to the solution and allowed to stir for 1 minute. IOOuL of 0.03M hydroquinone was added to the solution and allowed to stir for approximately 1 minute until the color stopped changing. 25uL of 34mM sodium citrate was added to the solution and allowed to stir for 2 minutes. The tube was capped and allowed to stir vigorously for 1 hour at room temperature. After 1 hour, the magnetic stir bar was removed from the tube, the tube was recapped, and the core-shell particles were centrifuged at 4000rpm for 20 minutes. The supernatant was removed without disturbing the pellet of particles, and the particles were resuspended in ImL of RNase free water. If necessary, the tube was sonicated briefly to break up the particle pellet. Nanoparticle Functionalization

[0094] The core-shell-shell nanoparticles were functionalized with miRNA mimics via the furan-based Diels- Alder linker and an NHS:EDC coupling. The ImL of core-shell-shell particles was immediately added to 7.5mL of the pre-prepared FDA linker in a glass scintillation vial. The vial was wrapped with parafilm and aluminum foil to block light. The vial was placed on a rocker at room temperature overnight to form the thiol linkage to the gold shell of the particle. Briefly, the solution was mixed and transferred to a 50mL conical centrifuge tube. The solution was centrifuged at 4000rpm for 20 minutes. The supernatant was removed, and the particles were resuspended in lOmL isopropanol to wash. The solution was centrifuged at 4000rpm for 20 minutes. The isopropanol wash was repeated 2 more times for a total of 3 isopropanol washes. Then, one wash with lOmL RNase free water was performed. The solution was centrifuged at 4000rpm for 20 minutes. The supernatant was removed, and the particles were resuspended in ImL RNase free water. IOOUL of lOOmM NHS:EDC at a 1:1 molar ratio was added to the solution. 25uL of the desired lOpM miRNA mimic, which was functionalized by IDT with an amine group, was added to the NHS:EDC, nanoparticle solution. For the combinatorial group at a ratio of 90%: 10%, 22.5uL of the lOpM miR-34a-5p mimic and 2.5uL of the lOpM miR-148b-3p mimic were added. The solution was pipetted to mix, wrapped in aluminum foil to block the light, and placed on the rocker overnight at room temperature to allow for covalent binding. Finally, the solution was centrifuged at 4000rpm for 20 minutes, the supernatant was removed, and the nanoparticle pellet was resuspended for downstream applications.

[0095] Nanoparticle Characterization

[0096] Nanoparticles were characterized after each stage of synthesis using dynamic light scattering. Briefly, nanoparticles were resuspended in ImL of RNase free water. An additional 2mL of RNase free water was added to the cuvette so that the solution reached reading height. The nanoparticles were transferred to a compatible cuvette and measured with the Malvern Zetasizer Nano ZS instrument. After each stage, the UV-vis spectrum of the nanoparticles was also measured. The nanoparticles were resuspended in ImL of RNase free water and transferred to a compatible cuvette. An additional 2mL of RNase free water was added to the cuvette so that the solution reached reading height. The particles were then measured on the Agilent Cary 5000 UV-Vis spectrophotometer from 400-900nm. For TEM and STEM-EDX characterization, nanoparticles were resuspended in ethanol. Alacey carbon 200 mesh copper grid was secured in tweezers and 2uL of nanoparticles solution was added to the grid. The grid was allowed to dry overnight. Finally, the nanoparticles were imaged on the FEI Talos F200X for TEM and STEM-EDX.

[0097] Photothermal Release of miRNA Mimics

[0098] Core-shell-shell nanoparticles were synthesized and functionalized with 90% miR-34a-5p and 10% miR-148b-3p miRNA mimics via the furan-based DA linker per the standard methods described here. The miR-34a-5p mimic was modified by IDT to contain FAM on the 3’ terminus. The miR-148b-3p was modified by IDT to contain Cy5 on the 3’ terminus.

[0099] Nanoparticles were centrifuged at 4000xg for 10 minutes and the supernatant was removed. The nanoparticles were then resuspended in 200uL of RNase free water and moved into a 96-well plate. For each well of the plate (n=l), ImL of CSS nanoparticles was prepared. An external stimulus was applied to n=3 wells per group for the following groups: 60 seconds of nIR irradiation, 90 seconds of nIR irradiation, 120 seconds of nIR irradiation, and 10 minutes at 100°C in an oven. Following external activation, the samples were moved from the well plate to individual microcentrifuge tubes and centrifuged at 4000xg for 10 minutes. The supernatant from each tube was removed and placed into a well on an opaque 96-well plate. A standard curve of miRNA was prepared by a two-fold serial dilution. Fluorescence for FAM and Cy5 was measured on a SpectraMax iD3 microplate reader.

[0100] Cell Culture

[0101] FaDu cells were purchased from ATCC and received at passage 120. They were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% Fetal Bovine Serum and 1% Penicillin / Streptomycin in an incubator at 37°C and 5% CO2. Cells were passaged as needed with trypsin / EDTA.

[0102] Cell Transfection via Lipofectamine RNA iMAX

[0103] Lipofectamine RNA iMAX was used for preliminary studies to determine which miRNA mimics and ratios were effective in treating FaDu cells. Upon receiving Lipofectamine RNA iMAX, a quick optimization assay was performed to limit batch to batch variation in transfection efficiency. Cells were seeded at 10,000 cells / well in a 96-well plate and incubated overnight. The following day, varying concentrations of lipofectamine were added to each well and allowed to incubate overnight. At the 24-hour timepoint, LIVE / DEAD staining was used to quantitatively determine the highest amount of lipofectamine with the lowest amount of resulting cell death. Images were captured using an Olympus 1X73 inverted fluorescent microscope. A fluorescence measurement via a SpectraMax iD3 microplate reader was used to confirm the accuracy of the LIVE / DEAD images. This optimized concentration of lipofectamine was used for all downstream assays. The remainder of the Lipofectamine RNA iMAX usage was conducted according to manufacturer protocol. For delivery of miRNA mimics with Lipofectamine RNA iMAX, cells were seeded at 10,000 cells / well in a 96-well plate and incubated overnight. The next day, lipofectamine-miRNA complexes were formed according to the described procedure in OPTLMEM and added to the cells. Cells were incubated for various timepoints and used in downstream assays.

[0104] Cell Transfection via Core-Shell-!

[0105]

[0106] For delivery of miRNA mimics via core-shell-shell nanoparticles, cells were seeded at 10,000 cells / well in a 96-well plate and incubated overnight. The next day, the nanoparticles were centrifuged at 4000xg and resuspended in OPTLMEM. The nanoparticles in medium were added to the cells at the desired concentration and incubated for 2 hours. Cells were then irradiated with nIR light using an 850nm LED from ThorLabs for 90 seconds per well at a maximum of 1cm from the bottom of the plate. Cells were incubated for various timepoints and used in downstream assays.

[0107] Intracellular Uptake via Confocal Microscopy

[0108] Cells were seeded in a glass bottom cell culture dish with 4 compartments at 75,000 cell s / compartm ent and incubated overnight. The next day, the nanoparticles were centrifuged at 4000xg and resuspended in OPTLMEM. Nanoparticles were functionalized with miRNA mimics tagged with a fluorophore. The miR-34a-5p mimic was modified by IDT to contain FAM on the 3’ terminus. The miR-148b-3p was modified by IDT to contain Cy5 on the 3’ terminus. For each compartment, 1.5mL of the nanoparticles was added to visualize uptake. Cells and nanoparticles were incubated together for 2 hours and then irradiated with nIR light using an 850nm LED from ThorLabs for 90 seconds per compartment at a maximum of 1cm from the bottom of the dish. The cells were then washed lx with PBS- at room temperature and lightly fixed with 4% paraformaldehyde for 4 minutes. After light fixation, the cells were washed 2x with PBS- and stained with DAPI at lug / mL in PBS- for 15 minutes at 37°C. After DAPI staining, cells were washed 2x with PBS- and resuspended in a small volume of PBS- to cover the bottom of the dish while imaging. Cells were imaged using the Zeiss LSM 880 Airyscan Fast with PicoQuant FLIM sampled at Nyquist. Lasers lines at 405nm, 488nm, and 633nm were used for DAPI, FAM, and Cy5 respectively.

[0109] Cell Viability

[0110] Cell viability was tested for cells treated with miRNA mimics via Lipofectamine RNA iMAX and core-shell-shell nanoparticles. Cell transfection methods were followed as describe above. For assays with delivery via lipofectamine RNAiMAX, each well contained a lOOnM concentration of miRNA mimic. For assays with delivery via core-shell-shell nanoparticles, each well contained a 50nM concentration of miRNA mimic. On the left half of the plate, control groups were not irradiated with nIR light from the 850nm LED. On the right side of the plate, all groups were irradiated with nIR light from the 850nm LED. Two assays were utilized to determine cell viability at 24, 48, and 72 hours: the LIVE / DEAD Viability / Cytotoxicity and the Quant IT PicoGreen dsDNA assay. The LIVE / DEAD Viability / Cytotoxicity kit was used according to manufacturer protocol. Images were captured using an Olympus 1X73 inverted fluorescent microscope. At each timepoint, the cell plate for the PicoGreen assay was frozen at -80°C. Once plates for all timepoints were frozen, the PicoGreen assay was performed according to manufacturer protocol and compared to a standard curve plate. A fluorescence measurement via a SpectraMax iD3 microplate reader was used to confirm the accuracy of the LIVE / DEAD images and to collect the PicoGreen data.

[0111] Bioinformatic Analysis

[0112] Bioinformatic analysis was performed in the QIAGEN Ingenuity Pathway Analysis (IPA) program. The program was opened, and a new project was formed. A specific miRNA, either miR-34a-5p or miR-148b-3p, was searched and added to a new “My Pathway.” Once this pathway was formed, the grow functionality was used to discover targets of the miRNA from the IPA database. Conditions for the targets included indirect or direct targets, miRNA to messenger RNA interactions, and a confidence level of experimentally observed or highly predicted. These settings were applied to the miRNA and run to form a network of targets. Once the targets were established, a new “My Pathway” was formed and the whole miRNA to target network was copied into it. Next the “Overlay Canonical Pathways” function was used to explore which targets were members of key cellular signaling pathways, including the apoptosis signaling pathway. Once the canonical pathway was overlayed, all miRNA targets not included in the pathway were deleted from the file. Next, the grow function was used again. This time it identified protein-protein interactions between the targets of the miRNA themselves. Conditions for these relationships included experimentally observed, direct or indirect connections of all interactions. The interactions were added to the “My Pathway.” Next, the “My Pathway” was opened into “Path Designer” for final formatting. Cell shapes were added to the figure for more context. The miRNA and targets were dragged to the level of the cell in which they typically function, such as the nucleus, the cytoplasm, the cell membrane, or outside of the cell. Depending on the complexity of the figure, the miRNAto messenger RNA interactions were artificially colored in blue while the protein-protein interactions were colored in green. A legend was added to the figure for clarity. All files were saved, and the cellular view of the final results was exported for use.

[0113] Subsequently, TargetScan v8.0, an online database maintained by the Whitehead Institute, was used to determine the predicted occupancy of miRNA to targets as identified by the QIAGEN IPA analysis. On the TargetScan website, a search was generated for each specific miRNA in humans. The full table was downloaded to the excel format. The genes identified in IPA were found and the occupancy data was exported to a smaller table for comparison.

[0114]

[0115] To study gene expression after miRNA treatment to FaDu cells, cells were transfected with Lipofectamine RNAiMAX as described above. Cells were seeded at 150,000 cells / well in a 12 well plate. The large RNA was isolated from the cells at 3-, 6-, 9-, and 12-hour timepoints post transfection using common isolation reagents such as Trizol and chloroform. The isolated RNA was then purified with diluted ethanol via a spin-column, resuspended in RNase free water, measured with a nanodrop, and frozen at -80°C until downstream use. A Verso cDNA synthesis kit was used according to manufacturer protocol to convert the RNA to complimentary DNA using a QuantStudio 3 thermocycler. The cDNA was measured using the nanodrop and diluted for use in RT-qPCR reactions. Primers for RT-qPCR were selected from PrimerBank, an online database developed at Harvard University, and purchased from IDT.23'25APowerUp Sybr master mix was used with the primers and cDNA according to manufacturer protocol to run the RT-qPCR on the QuantStudio 3 thermocycler. For each well, 20ng of cDNA was used. The data was exported to Excel via the Design and Analysis software.

[0116] Animal Studies

[0117] The studies were conducted according to the US Department of Health and Human Services' Guide for the Care and Use of Laboratory Animals. All animal studies are approved under Penn State IACUC protocol PRAMS201747826. Equal numbers of adult male and female mice were used in each group with Foxnl<nu> mice. Two studies were conducted congruently, a short-term efficacy test and a long-term efficacy test. For both tests, 5 million FaDu cells at a 1:1 ratio with Matrigel were injected subcutaneously into the left and right flank of each mouse while it was anesthetized with 5% isoflurane and maintained at 2-3% isoflurane. The tumors were allowed to grow until palpable, approximately one week, and were then measured by caliper. When the tumors reached between 0.3-0.8cm2, the mice were randomly divided into 5 groups: No treatment which received a saline injection, CSS miR-NC which had a randomized “non-coding” miRN A attached, CSS miR-34a, CSS miR-148b, or CSS miR-34a and miR-148b (90: 10) which is the combinatorial group. At this tumor size, the mice received a retro-orbital injection of the therapeutic for the specified group. For all groups containing miRNA attached to the CSS nanoparticles, 100 picomoles of miRNA was delivered. For the short-term study, the left tumor was not treated with near-infrared light to serve as a control without photo-activation where the therapeutic should not be released. Starting 6 hours after the retro-orbital injection of the therapeutic, the right tumor was irradiated with near-infrared light for 5 minutes using a ThorLabs 850nm 900mW mounted LED while mice were anesthetized as previously described and on a heating pad. For the long-term study, both the left and right tumor were irradiated with near-infrared light for 5 minutes using a ThorLabs 850nm 900mW mounted LED while mice were anesthetized as previously described and on a heating pad. For the next 7 days, the mice were monitored daily, weighed, and measured via caliper to determine tumor volume. Seven days after the therapeutic injection and photo-activation, the mice from the short-term study were euthanized with CO2 followed by cervical dislocation. Tissue samples were collected and stored in 10% neutral buffered formalin for histological analysis of samples. Following day 7, the long-term study continued. Mice were monitored frequently based on tumor volume and according to the IACUC protocol. For the long-term study, the mice were euthanized at humane endpoints established in the IACUC protocol. Tissue samples were collected and stored in 10% neutral buffered formalin.

[0118] Histology Analysis

[0119] Tissue samples were incubated in 10% neutral buffered formalin overnight. Tissues were washed with 70% ethanol for 15 minutes and then sliced into 4mm thick sections.

[0120] Following slicing, the tissue processing protocol from Leica was used to prepare samples. Dehydration included 6 steps: 15 minutes in 70% ethanol, 15 minutes in 90% ethanol, 15 minutes in 100% ethanol, 15 minutes in 100% ethanol, 30 minutes in 100% ethanol, and 45 minutes in 100% ethanol. Clearing in xylene substitute was completed in 3 steps of 20 minutes, 20 minutes, and 45 minutes sequentially. Wax infiltration was performed between 56-60°C in a compact oven for 30 minutes, 30 minutes and 45 minutes sequentially. The sample was then embedded in wax using a Leica EG1150. Samples were sliced via microtome in 10pm sections, placed into a water bath, and lifted onto a glass slide. Tissue sections were dewaxed and stained using a Leica ST5010 Autostainer XL. Samples were mounted using a xylene-based mounting media. Completed samples were imaged with the Olympus 1X73 inverted fluorescent microscope.

[0121] Statistical Analysis

[0122] GraphPad Prism 10 was used to graph data and perform the statistical analysis.

[0123] ANOVA analysis was used to compare groups within each experiment. The statistical significance and sample size for each experiment are located within respective igure description. The results are shown as the mean ± the SD.

[0124] Data obtained using the materials and methods described above is summarized by way of the Figures.

[0125] While various examples of the present disclosure are described herein, it should be understood that they have been presented by way of example only, and not limitation. It will be apparent to persons skilled in the relevant art that various changes in form and detail can be made therein without departing from the spirit and scope of the present disclosure. Thus, the breadth and scope of the present disclosure should not be limited by any of the exemplary examples provided herein.

Claims

What is claimed is:

1. A method for treating prostate cancer in an individual in need thereof, the method comprising administering to the individual a combination of miR-34a-5p or mimic thereof and miR-222-3p or mimic thereof in a 50:50 molar ratio, and optionally in a 90:10 molar ratio, respectively.

2. The method of claim 1, wherein the miR-34a-5p or the mimic thereof and the miR-222-3p or the mimic thereof are conjugated to the same nanoparticle in the 90: 10 molar ratio, and wherein a plurality of the nanoparticles are administered to the individual.

3. The method of claim 2, wherein stimulation of the nanoparticles with light releases the miRNAs from the nanoparticles to thereby treat the cancer.

4. The method of claim 3, wherein the miR-34a-5p mimic and the miR-222-3p are used and wherein the miR-34a-5p mimic comprises the sequence of SEQ ID NO:2 and the mir-222-3p mimic comprises the sequence of SEQ ID NO:4.

5. A method for treating an individual for head and neck cancer, the method comprising administering to the individual a combination of miR-148b-3p or a mimic thereof and miR-34a-5p or a mimic thereof at a 50:50 molar ratio, and optionally in a 10:90 ratio, respectively.

6. The method of claim 5, wherein the miR-148b-3p or a mimic thereof and the miR-34a-5p or the mimic thereof are conjugated to the same nanoparticle in the 10:90 molar ratio, and wherein a plurality of the nanoparticles are administered to the individual.

7. The method of claim 6, wherein stimulation of the nanoparticles with light releases the miRNAs from the nanoparticles to thereby treat the cancer.

8. The method of claim 7, wherein the hsa-miR-148b-3p mimic and the miR-34a-5p mimic are used, and wherein the miR-148b-3p mimic comprises the sequence of SEQ ID NO:3 and the miR-34a-5p mimic comprises the sequence of SEQ ID NO:2.

9. A method for treating an individual for bladder cancer, the method comprising administering to the individual a combination miR-3619-5p or a mimic thereof and miR-26b-5p or a mimic thereof in a 50:50 molar ratio or optionally a 90: 10 ratio, respectively.

10. The method of claim 9, wherein the miR-3619-5p or the mimic thereof and the miR-26b-5p or the mimic thereof are conjugated to the same nanoparticle in the 50:50 ratio, and wherein a plurality of the nanoparticles are administered to the individual.

11. The method of claim 10, wherein stimulation of the nanoparticles with light releases the miRNAs from the nanoparticles to thereby treat the cancer.

12. The method of claim 11, wherein the miR-3619-5p mimic comprises the sequence of SEQ ID NO:5 and the miR-26b-5p mimic comprises the sequence of SEQ ID NO:6.

13. A plurality of nanoparticles wherein each nanoparticle in the plurality of nanoparticles comprises an miR-34a-5p or mimic thereof and an miR-222-3p or mimic thereof conjugated to each nanoparticle in the plurality of nanoparticles in a 90:10 molar ratio, respectively.

14. A plurality of nanoparticles wherein each nanoparticle in the plurality of nanoparticles comprises miR-148b-3p or a mimic thereof and an miR-34a-5p or a mimic thereof conjugated to each nanoparticle the plurality of nanoparticles at a 10:90 molar ratio, respectively.

15. A plurality of nanoparticles wherein each nanoparticle in the plurality of nanoparticles comprises miR-3619-5p or a mimic thereof and miR-26b-5p or a mimic thereof conjugated to each nanoparticle the plurality of nanoparticles at a 50:50 molar ratio, respectively.

Images