Fluorescent nanodiamond for image guided targeted drug delivery

Abstract

The subject invention provides materials and methods for treating diseases affecting the central nervous system (CNS) and / or other viral reservoir organs utilizing nanoscopic diamond particles, i.e., nanodiamonds (ND), loaded with drug molecules and microglial targeting moiety.

Description

FJ ref. UTRGV-P0032WO / Client ref. 2024-0009FLUORESCENT NANODIAMOND FOR IMAGE GUIDED TARGETED DRUG DELIVERYRELATED APPLICATIONS

[0001] This Application is an International Application claiming priority to US Provisional Patent applications 63 / 697,460 filed 9 / 21 / 2024, each of which is incorporated herein by reference in their entirety.STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

[0002] This invention was made with government support under Grantl4140609 awarded by the National Institutes of Health. The government has certain rights in the invention.REFERENCE TO SEQUENCE LISTING

[0003] A sequence listing required by 37 CFR 1.821-1.825 is being submitted electronically with this application. The sequence listing is incorporated herein by reference. The sequence listing that is contained in the file named "UTRGVP0032" which is 3,880 bytes (as measured in Microsoft Windows®) and was created on 9 / 20 / 2025.BACKGROUND

[0004] Nanotechnology has emerged as a transformative field with vast potential across various industries, including biomedical applications. Among these, nanodiamonds (NDs) have garnered significant attention due to their unique properties, such as biocompatibility, chemical stability, and tunable surface functionalities. These properties make NDs promising candidates for therapeutic interventions in biomedicine.

[0005] Nanodiamonds are typically synthesized through techniques such as detonation, chemical vapor deposition, or milling, resulting in nanoscale particles with diameters ranging from a few to several hundred nanometers. Their crystalline structure, composed primarily of sp3-bonded carbon atoms arranged in diamond cubic lattice, endows NDs with exceptional mechanical hardness and stability, crucial for enduring harsh biological environments.

[0006] In biomedical research, the application of NDs spans a wide range of therapeutic modalities. One of the prominent areas of exploration involves drug delivery systems. NDs can serve as carriers for various drugs and biomolecules due to their high surface area and the ability to functionalize their surfaces with targeting moieties. This capability enables precise delivery ofFJ ref. UTRGV-P0032WO / Client ref. 2024-0009 therapeutic agents to specific cells or tissues, thereby minimizing off-target effects and enhancing therapeutic efficacy. Previous patented technology US 9,616,022 has established the potential of NDs as a targeted vehicle for the brain. However, it is still difficult to track the efficacy of drug delivery on the central nervous system (CNS), such as the capacity to navigate. Without knowing if or how much of the drug is reaching the target area, it is hard to optimize dosage or confirm that a treatment is working.

[0007] Moreover, some of the nanodiamonds exhibit intrinsic fluorescent properties, stemming from nitrogen-vacancy (NV) centers within their structure. This fluorescence can be leveraged for bioimaging applications, allowing for real-time tracking of ND-based drug carriers or for visualizing biological processes at the nanoscale level.

[0008] Beyond drug delivery and imaging, nanodiamonds have shown promise in other therapeutic approaches. They possess excellent biocompatibility, which is crucial for their integration into biological systems without eliciting adverse immune responses or cytotoxic effects. Additionally, NDs have been explored for their potential in regenerative medicine, as scaffolds for tissue engineering, and as agents for photothermal therapy due to their ability to convert light energy into heat.

[0009] However, despite their promising potential, challenges remain in the large-scale synthesis of uniform NDs with precise control over their size, surface chemistry, and functionalization. Furthermore, the long-term biocompatibility and biodegradability of NDs in vivo need further investigation to ensure their safe clinical translation.

[0010] HIV, neuroinflammation, and combined antiretroviral therapy (cART): cART significantly improves the mean lifespan and quality of life but HIV persists in reservoir organs like the brain. Moreover, several cART drugs have shown limitations in distribution and penetration into the brain. This commonly results in other complications, such as HIV-associated neuroinflammation and neurodegeneration. These pathologies are the most common manifestation of HIV disease that causes cognitive impairment with no further treatment options. Thus, research towards developing new anti-retroviral medicines to improve drug delivery to the brain remains active. These drugs need to be reformulated so that they can effectively cross the BBB and ensure their delivery to the microglia.

[0011] Nanomedicine-based drug delivery with M2NDs. Nanomedicine has introduced several options for targeted drug delivery in the brain. Prior research has established thatFJ ref. UTRGV-P0032WO / Client ref. 2024-0009 macrophages are potential drug vehicles for the transport and delivery of nanodrugs in the brain. The inventor has demonstrated that cells pretreated with the nanodrug can perform sustained drug release in the brain. In this regard, previous studies have demonstrated ND’s capability as a drug carrier of doxorubicin, purvalanol A, 4-hydroxytamoxifen, and dexamethasone for the treatment of colon cancer, liver cancer, breast cancer, and blood cancer, respectively. Furthermore, these studies also showed its drug loading and sustained release capacity without producing any inflammatory reaction in human cells. Previous studies indicated that ND is not toxic to many different human cell types. Furthermore, ND has distinct advantages over other carbon-based nanomaterials such as carbon nanotubes and nanographene, which already showed toxicity and limited dispersity in water, making it difficult to prepare nanodrugs with these excipients.SUMMARY

[0012] One solution to current problems related to NDs and CNS treatments is based on pharmaceutical compositions comprising a plurality of nanodiamond (ND) particles, each ND particle having a surface with drug molecules and microglial targeting moiety absorbed thereon. One composition is a multipurpose next-generation ND (M2ND), which will have a sub- 100 nm size range, a surface modified for biologically active molecule attachment, higher colloidal stability, adjustable zeta potential, and enhanced capabilities of intrinsic fluorescence due to the incorporation of specific atomic-scale crystal lattice defects known as the nitrogen-vacancy center (NV center). M2ND can possess remarkable fluorescent properties and photostability and thereby can be used as an optical probe in the CNS. This new generation M2ND can be instrumental as a multifunctional, CNS-trackable delivery vehicle in a single nanostructure. Nanoformulations will have an improved distribution to HIV reservoirs in an efficient, safe, and site-specific manner. One embodiment is M2ND-mediated tracking of microglia in the brain tissues and delivery of anti-HIV drugs or other therapeutics. M2ND can be conjugated with three clinically available cART drugs and further conjugated with microglia-specific antibody Tmeml l9 for target-specific tracking and delivery. M2ND can be taken up by the circulating monocytes entering the brain and crossing the BBB in response to neuroinflammation. M2ND can be released from the monocytes and selectively attached to microglia with a specific Tmeml l9 surface receptor. The surfaces of the ND particles are modified with at least oneFJ ref. UTRGV-P0032WO / Client ref. 2024-0009 functional group selected from carboxyls, lactones, ketones, ethers, hydroxyls, and amines. The drug molecules can be selected from dolutegravir, tenofovir, and emtricitabine, or other drugs of similar function. The pharmaceutical composition is between 10, 20, 30, 40, 50, 60, 70, 80, 90, to 100 nm in diameter (including all values and ranges there between) and can pass through the blood brain barrier of a subject. The capability of the pharmaceutical composition reaching a target area can be observed via ELISA, X-ray diffraction, photoluminescence spectroscopy, or ultra-fast transmission electron microscopy. The surface electrostatic potential of ND makes it possible to attract water molecules and efficiently absorb drug molecules. The pharmaceutical composition can be tracked from the site of administration until it reaches the brain where M2ND exhibits a sustained release of the drug molecule due to its reversible surface electrostatic potential. This process helps to detach the drug molecule through the desorption mechanism. The composition can also be tracked from the site of administration to the delivery site through a ODMR technique, due to natural fluorescent color centers of NDs. Nanoformulations will have an improved distribution to HIV reservoirs in an efficient, safe, and site-specific manner.

[0013] Developing treatment for HIV infection in the brain is different from any other brain- related disease because HIV is present in many different cells of brain tissues. Whole brain- targeted therapy is generally not helpful because these different cells need different dosing to reduce / eliminate viral loads.

[0014] This application describes advancements of the current technology with two innovative approaches to deliver drugs to the microglia. Primarily, the formulation is nextgeneration, reasonably small (50-100 nm or less) with fluorescence capability. The molecular design also includes clinically approved anti-HIV drugs and a unique microglial marker Tmeml l9 on the surface. This M2ND has proven to be safe for human cells, making this formulation superior to other formulations. Secondly, the capability of tracking the drugs from the site of administration to the delivery site through a novel ODMR technology, makes this formulation very unique compared to any other existing nano drug. New application: The proposed M2ND formulation will target active inflammatory areas of the brain tissue, where most of the infected microglia are present. Thus, our M2ND-based targeting strategy for microglia is a novel, paradigm-changing approach with a fluorescence-based image-guided drug delivery strategy.FJ ref. UTRGV-P0032WO / Client ref. 2024-0009

[0015] Certain embodiments are directed to a nanodiamond composition comprising a microglial targeting moiety and an anti-HIV drug operatively coupled to a nanodiamond particle. A microglial targeting moiety refers to a molecule or compound that interacts with or binds to microglia, which are the immune cells of the brain. The term moiety as used herein refers to part of a composition having a define purpose or characteristic, such as a binding moiety which can be proteins, carbohydrates, lipids, etc. In certain aspects a microglial targeting moiety can be Tmeml l9, PDGF0, fragments thereof. The nanodiamond particle can be modified with at least one, or one or more functional group selected from carboxyls, lactones, ketones, ethers, hydroxyls, and / or amines. In certain aspects an anti-HIV drug can be dolutegravir, tenofovir, emtricitabine, lamivudine, abacavir, zidovudine, efavirenz, rilpivirine, doravirine, atazanavir, darunavir, lopinavir / ritonavir, bictegravir, raltegravir, elvitegravir, enfuvirtide, maraviroc, lenacapravir or other drugs of similar function. In certain aspects the nanodiamond composition is between 50-100 nm in diameter. The nanodiamond particle can be detected using ELISA, X- ray diffraction, photoluminescence spectroscopy, ultra-fast transmission electron microscopy, or other appropriate methods or detectors. The nanodiamond particle can have a reversible surface electrostatic charge.

[0016] Antiretroviral drugs used to treat HIV infection work by targeting different stages of the virus's life cycle, helping to suppress viral replication, maintain immune function, and prevent progression to AIDS. These drugs are typically used in combination regimens, known as antiretroviral therapy (ART), to maximize effectiveness and minimize resistance. Nucleoside / nucleotide reverse transcriptase inhibitors (NRTIs), such as tenofovir, emtricitabine, lamivudine, abacavir, and zidovudine, block the reverse transcriptase enzyme, preventing HIV from converting its RNA into DNA. Non-nucleoside reverse transcriptase inhibitors (NNRTIs), including efavirenz, rilpivirine, doravirine, and nevirapine, bind directly to reverse transcriptase to inhibit its function. Protease inhibitors (Pls), such as atazanavir, darunavir, lopinavir, and ritonavir (often used as a booster), interfere with the protease enzyme, which is needed to produce mature viral particles. Integrase strand transfer inhibitors (INSTIs), like dolutegravir, raltegravir, bictegravir, and elvitegravir, prevent HIV from integrating its DNA into the host cell’s genome. Entry inhibitors, such as enfuvirtide (a fusion inhibitor) and maraviroc (a CCR5 antagonist), block HIV from entering host cells by targeting viral or cellular proteins involved in attachment and entry. Pharmacokinetic enhancers, like cobicistat, are used to boost the levels ofFJ ref. UTRGV-P0032WO / Client ref. 2024-0009 other antiretrovirals, improving their efficacy. Fixed-dose combinations, such as Biktarvy (bictegravir / emtricitabine / tenofovir alafenamide) or Triumeq (dolutegravir / abacavir / lamivudine), combine multiple drugs into a single pill for simplified dosing. Each class targets a unique aspect of HIV replication, and regimens are tailored based on patient factors like viral load, resistance patterns, and tolerability.

[0017] Other embodiments are directed to methods for treating HIV infection, comprising administering a nanodiamond composition comprising a microglial targeting moiety and an antiHIV drug operatively coupled to a nanodiamond particle. The microglial targeting moiety can be Tmeml 19 or PDGFp. In certain methods the nanodiamond particle is modified with at least one, or one or more functional groups including but not limited to carboxyls, lactones, ketones, ethers, hydroxyls, and / or amines. The anti-HIV drug can be one or more of dolutegravir, tenofovir, and emtricitabine, or drugs with similar function. The method can use a nanodiamond composition is between 50-100 nm in diameter. In certain aspects the method can include detecting or locating the nanodiamond composition using ELISA, X-ray diffraction, photoluminescence spectroscopy, or ultra-fast transmission electron microscopy. The method can use a nanodiamond particle that has a reversible surface electrostatic charge.

[0018] Other embodiments of the invention are discussed throughout this application. Any embodiment discussed with respect to one aspect of the invention applies to other aspects of the invention as well and vice versa. Each embodiment described herein is understood to be embodiments of the invention that are applicable to all aspects of the invention. It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions and kits of the invention can be used to achieve methods of the invention.

[0019] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0020] Throughout this application, the term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0021] The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.”FJ ref. UTRGV-P0032WO / Client ref. 2024-0009

[0022] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0023] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains”, “containing,” “characterized by” or any other variation thereof, are intended to encompass a non-exclusive inclusion, subject to any limitation explicitly indicated otherwise, of the recited components. For example, a chemical composition and / or method that “comprises” a list of elements (e.g., components or features or steps) is not necessarily limited to only those elements (or components or features or steps), but may include other elements (or components or features or steps) not expressly listed or inherent to the chemical composition and / or method.

[0024] As used herein, the transitional phrases “consists of’ and “consisting of’ exclude any element, step, or component not specified. For example, “consists of’ or “consisting of’ used in a claim would limit the claim to the components, materials or steps specifically recited in the claim except for impurities ordinarily associated therewith (i.e., impurities within a given component). When the phrase “consists of’ or “consisting of’ appears in a clause of the body of a claim, rather than immediately following the preamble, the phrase “consists of’ or “consisting of’ limits only the elements (or components or steps) set forth in that clause; other elements (or components) are not excluded from the claim as a whole.

[0025] As used herein, the transitional phrases “consists essentially of’ and “consisting essentially of’ are used to define a chemical composition and / or method that includes materials, steps, features, components, or elements, in addition to those literally disclosed, provided that these additional materials, steps, features, components, or elements do not materially affect the basic and novel characteristic(s) of the claimed invention. The term “consisting essentially of’ occupies a middle ground between “comprising” and “consisting of’.

[0026] As used herein the term "conjugate" means to connect two compounds through formation of one or multiple covalent and / or non-covalent interactions. In certain embodiments, the one or multiple covalent or non-covalent interactions may be cleavable at a physiological condition of a subject. Examples of such cleavable interactions include, without limitation,FJ ref. UTRGV-P0032WO / Client ref. 2024-0009 cleavable covalent bonds such as bisulfide, ether, ester, amide, thio-ether, thio-ester, carbonate, carbamate, phosphate, and oxime bonds, and non-covalent interactions.

[0027] The examples and embodiments described herein are for illustrative purposes only and various modifications or changes in light thereof will be suggested to persons skilled in the art and are included within the spirit and purview of this application. In addition, any elements or limitations of any invention or embodiment thereof disclosed herein can be combined with any and / or all other elements or limitations (individually or in any combination) or any other invention or embodiment thereof disclosed herein, and all such combinations are contemplated with the scope of the invention without limitation thereto.

[0028] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0029] Definitions - The following terms, as used in this specification and the appended claims, have the meanings set forth below. These definitions are provided to ensure clarity and consistency in understanding the scope and intent of the invention. Unless otherwise indicated, these terms shall apply throughout the application.

[0030] Anti-HIV drug: Refers to therapeutic agents for treating HIV, including but not limited to dolutegravir, tenofovir, emtricitabine, or other drugs of similar function (e.g., Nucleoside Reverse Transcriptase Inhibitors (NRTIs) such as Lamivudina (LMV), Emtricitabina (FTC), Zidovudina (ZDV), Tenofovir (TDF), or Abacavir (ABC); Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) such as Nevirapine, Efavirenz, Etravirine, or Rilpivirine; Protease Inhibitors such as Amprenavir, Atazanavir, Darunavir, Fosamprenavir, Indinavir, Lopinavir, Nelfinavir, Ritonavir, Saquinavir, or Tipranavir; Integrase Strand Transfer Inhibitors (INSTIs) such as Raltegravir, Elvitegravir, Dolutegravir (DTG), or Bictegravir; Fusion Inhibitors such as Enfuvirtide; CCR5 Antagonists such as Maraviroc; Capsid Inhibitors such as Lenacapavir (Sunlenca); and Post-Attachment Inhibitors such as Ibalizumab).

[0031] Blood-Brain Barrier (BBB): The selective barrier formed by endothelial cells, astrocytes, and pericytes that restricts the passage of substances from the bloodstream into theFJ ref. UTRGV-P0032WO / Client ref. 2024-0009 brain, protecting the central nervous system (CNS) while posing a challenge for drug delivery. In the context of this invention, the BBB is a critical obstacle that the nanodiamond composition (M2ND) is designed to cross to deliver anti-HIV drugs to microglia.

[0032] Colloidal Stability: The ability of nanodiamond particles, such as M2ND, to remain uniformly dispersed in a liquid medium without aggregating or settling, ensuring consistent performance in biological environments for drug delivery and tracking.

[0033] Conjugate: To connect two compounds through formation of one or multiple covalent and / or non-covalent interactions. In certain embodiments, the one or multiple covalent or non- covalent interactions may be cleavable at a physiological condition of a subject. Examples of such cleavable interactions include, without limitation, cleavable covalent bonds such as bisulfide, ether, ester, amide, thio-ether, thio-ester, carbonate, carbamate, phosphate, and oxime bonds, and non-covalent interactions.

[0034] Fluorescent nanodiamond (FND) particles: A specialized class of nanodiamonds that exhibit unique optical properties, typically ranging in size from a few to several tens of nanometers, characterized by their diamond-like carbon structure (sp3 -bonded carbon atoms in a cubic lattice). Their fluorescent properties arise primarily from defects within the diamond lattice, most notably nitrogen-vacancy (NV) centers, which create a quantum system with strong photoluminescence under excitation by green light and emission in the red to near-infrared spectral range. Features include biocompatibility, photostability, brightness and contrast, surface functionalization, and applications in bioimaging, drug delivery, sensing, and detection.

[0035] Microglia: The resident immune cells of the central nervous system (CNS), playing roles in immune surveillance, neuroinflammation, and synaptic maintenance.

[0036] Microglial targeting moiety: A molecule or compound that interacts with or binds to microglia, the immune cells of the brain. The term "moiety" refers to part of a composition having a defined purpose or characteristic, such as a binding moiety which can be proteins, carbohydrates, lipids, etc. Examples include Tmeml l9, PDGFb, fragments thereof; antibodies and antibody-derivatives (e.g., monoclonal antibodies to surface markers like CDl lb, CD45, or P2Y12); ligands and receptor agonists / antagonists (e g., for purinergic receptors like P2Y12 or toll-like receptors); cytokines and chemokines (e.g., CCL2 or IL-ip); or peptides and peptide- based agents.FJ ref. UTRGV-P0032WO / Client ref. 2024-0009

[0037] Moiety: Part of a composition having a defined purpose or characteristic, such as a binding moiety which can be proteins, carbohydrates, lipids, etc.

[0038] Multipurpose Next-generation Nanodiamond (M2ND): A next-generation trackable therapeutic molecule designed to target microglial cells in the brain for treating CNS disorders, particularly HIV infection. Attributes include: (1) a sub-100 nm size range to facilitate BBB penetration, (2) a surface modified for attachment of biologically active molecules, (3) high colloidal stability, (4) adjustable zeta potential for controlled drug release, and (5) enhanced intrinsic fluorescence due to nitrogen-vacancy (NV) centers, enabling real-time tracking via optically detected magnetic resonance (ODMR) and other imaging techniques. It can be conjugated with anti-HIV drugs (e.g., dolutegravir, tenofovir, emtricitabine) and microgliaspecific antibodies like Tmeml 19.

[0039] Nanodiamond (ND) particles: Nanoscale particles with diameters ranging from a few to several hundred nanometers, synthesized through techniques such as detonation, chemical vapor deposition, or milling. Composed primarily of sp3 -bonded carbon atoms in a diamond cubic lattice, endowing them with mechanical hardness, stability, biocompatibility, chemical stability, and tunable surface functionalities. Surfaces may include diamond or diamond-like carbons (e.g., graphite, fullerene, layered shells, amorphous carbon) and can be modified with functional groups like carboxyls, lactones, ketones, ethers, hydroxyls, and amines. Used as carriers for drugs and biomolecules, with intrinsic fluorescence from nitrogen-vacancy (NV) centers for bioimaging.

[0040] Nitrogen-vacancy (NV) centers: Crystallographic defects in nanodiamonds where a nitrogen atom replaces a carbon atom adjacent to a vacant lattice site, creating a quantum system with unique optical characteristics, including strong photoluminescence under green light excitation and fluorescence emission in the red to near-infrared range. Responsible for the fluorescent properties of FNDs.

[0041] Optically Detected Magnetic Resonance (ODMR): A double resonance technique used to initialize, manipulate, and read the electron spin state of crystal defects (e.g., nitrogenvacancy centers) in nanodiamonds, enabling optical tracking and visualization of M2ND in biological systems, such as microglia in the CNS.

[0042] PDGF beta (PDGF0 or PDGF-BB): One isoform of Platelet-Derived Growth Factor, a dimeric protein (BB chains) involved in cell growth, proliferation, and survival, particularly inFJ ref. UTRGV-P0032WO / Client ref. 2024-0009 mesenchymal cells like fibroblasts, smooth muscle cells, and glial cells. Can serve as a microglial targeting moiety.

[0043] Photoluminescence (PL): The emission of light from nanodiamond particles, particularly those with nitrogen-vacancy (NV) centers, following excitation by a light source (e.g., green light), enabling fluorescence-based imaging and tracking in biological applications.

[0044] Sustained Release: The controlled and gradual release of drug molecules (e.g., antiHIV drugs) from the nanodiamond particle’s surface, facilitated by its reversible surface electrostatic potential, allowing prolonged therapeutic effect in the target tissue (e.g., microglia in the CNS).

[0045] TMEM119 (Transmembrane Protein 119): A transmembrane protein predominantly expressed in microglia, serving as a specific cell-surface marker for microglia (not expressed in circulating monocytes, blood-derived macrophages, or other immune / neuronal cell types). Upregulated during HIV infection and cART treatment, making it suitable for targeted drug delivery. The precursor sequence is provided as SEQ ID NO: 1, with a signal peptide at amino acids 1-25, mature polypeptide at 26-283, and transmembrane region at 97-117. Can serve as a microglial targeting moiety.

[0046] Zeta Potential: The electrostatic potential at the surface of a nanodiamond particle, which can be adjusted to control drug adsorption and release, contributing to the stability and functionality of M2ND in biological environments.DESCRIPTION OF THE DRAWINGS

[0047] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of the specification embodiments presented herein.

[0048] FIG. 1A-1B shows the ND characterization. (A) Raman spectra of detonated NDs display the characteristic feature of the diamond phase at 1326 cm'1, graphitic carbon on ND surface at 1612 cm'1(broad) and smaller signal from surface hydroxyl groups. (Roy et.al, Scientific Reports, 2018); (B) XRD pattern (Mo Koc-radiation) of NDs include two peaks corresponding to diffraction from atomic planes with Miller indices (111) and (220) of the cubic diamond structure.FJ ref. UTRGV-P0032WO / Client ref. 2024-0009

[0049] FIG. 2 shows the cell viability of HPM cells after treatment with M2NDs. 50, 70, 100 nm respectively. The bars represent the mean percentage viability of HPM cells compared to the untreated controls, with the standard deviation indicated by the error bars.

[0050] FIG. 3 shows the intracellular ROS production in HPM cells with M2NDs.

[0051] FIG. 4A-4C shows the stability of M2ND in room temperature. (A) 50, (B) 70, and(C) 100 nm in PBS solution.

[0052] FIG. 5A-5B shows the immunocytochemical analysis of cellular uptake of M2NDs by HMC-3 cells. (A) shows HMC-3 cells after uptake of 50 nm FNDs. (B) depicts cells with 70 nm FNDs.

[0053] FIG. 6A-6B shows cell uptake of (A) M2ND-DTG and DTG in human PBMC, and (B) M2ND-FTC and FTC in human PBMC.

[0054] FIG. 7A-7C shows TMEM 119 expression in HMC-3 cells infected with HIV. (A) the RT-PCR results of TMEM expression in HMC-3 cells under different treatment conditions: control (untreated), HIV-infected, HIV-infected+cART (HIV+cART) and cART alone on day 5. (B) Protein expression through Western blot. The upper panel shows TMEM protein, and the lower panel displays P-actin. (C) The volumetric analysis of TMEM expression was significantly higher in HIV-infected cells and HIV+ cART treatment, with the y-axis representing relative expression. Error bars indicate the SEM denoting variability within replicates.

[0055] FIG. 8 shows antiretroviral efficacy study of M2ND formulations of TNF, DTG, and FTC compared to their respective free drugs.

[0056] FIG. 9A-9B shows the antiretroviral efficacy of M2ND nano-formulated TNF, DTG, and FTC compared to their respective FD in hu-PBL mice after 5 days of treatment. (A) HIV- LTR gene expression (PCR) represented with fold change compared to control. (B) Tmeml l9 gene expression (PCR) in HIV infected hu-PBL mice.DESCRIPTION

[0057] The following discussion is directed to various embodiments of the invention. The term “invention” is not intended to refer to any particular embodiment or otherwise limit the scope of the disclosure. Although one or more of these embodiments may be preferred, the embodiments disclosed should not be interpreted, or otherwise used, as limiting the scope of the disclosure, including the claims. In addition, one skilled in the art will understand that theFJ ref. UTRGV-P0032WO / Client ref. 2024-0009 following description has broad application, and the discussion of any embodiment is meant only to be an example of that embodiment and not intended to imply that the scope of the disclosure, including the claims, is limited to that embodiment.

[0058] Development of Multipurpose Next-Generation Nanodiamond (M2ND) for Targeted Drug Delivery. One objective of this invention is to develop a next-generation trackable therapeutic molecule, referred to as Multipurpose Next-generation Nanodiamond (M2ND), designed to target microglial cells in the brain for the treatment of central nervous system (CNS) disorders, particularly Human Immunodeficiency Virus (HIV) infection. This invention addresses the critical challenge of delivering therapeutic agents across the blood-brain barrier (BBB) to reach microglia, the resident immune cells of the CNS, which serve as a major HIV reservoir. M2ND is a novel nanodiamond-based platform with the following attributes: (1) a sub- 100 nm size range to facilitate BBB penetration, (2) a surface modified for attachment of biologically active molecules, (3) high colloidal stability, (4) adjustable zeta potential for controlled drug release, and (5) enhanced intrinsic fluorescence due to nitrogen-vacancy (NV) centers, enabling real-time tracking via optically detected magnetic resonance (ODMR) and other imaging techniques. These properties make M2ND a multifunctional, CNS-trackable drug delivery vehicle. The development of M2ND builds upon prior (see US 9,616,022), which established nanodiamonds as a targeted delivery vehicle for the brain, but left unresolved questions about their navigation mechanism from the administration site to microglial targets. To address these challenges, this invention employs a multidisciplinary approach, integrating expertise from neuroimmunology, materials science, and advanced imaging technologies.

[0059] The development and evaluation of M2ND are guided by three specific objectives: (1) Develop and characterize M2ND formulations with self-tracking capabilities, including stability, fluorescence, and toxicity assessments, and evaluate their target-specific tracking efficiencies using Tmeml 19 antibody conjugates; (2) Study the detection capability of M2ND in microglia using an in vitro BBB model with HIV-infected microglia to assess translocation and drug delivery, optimizing target-specific binding via ODMR technology; and (3) Evaluate the safety, tracking, and antiretroviral efficacy of M2ND in vivo using BALB / cJ and HIV-1 infected humanized mouse models, focusing on drug delivery to microglia and reduction of HIV infection. These objectives aim to establish M2ND as a non-invasive, trackable platform for delivering combination antiretroviral therapy (cART) drugs, such as dolutegravir, tenofovir, andFJ ref. UTRGV-P0032WO / Client ref. 2024-0009 emtricitabine, to microglia, leveraging the chronic neuroinflammation associated with HIV to facilitate monocyte-mediated transport across the BBB. This approach not only enhances drug delivery precision but also enables real-time monitoring of microglial targeting, offering significant advancements for treating HIV-associated neurocognitive disorders and potentially other CNS conditions.I. Fluorescent Nanodiamond Particles

[0060] Fluorescent nanodiamond (FND) particles are a specialized class of nanodiamonds that exhibit unique optical properties, making them valuable for a variety of biomedical and imaging applications. These nanodiamonds typically range in size from a few to several tens of nanometers and are characterized by their diamond-like carbon structure, consisting of sp3- bonded carbon atoms arranged in a cubic lattice.

[0061] The fluorescent properties of FNDs arise primarily from defects within the diamond lattice, most notably the nitrogen-vacancy (NV) centers. NV centers are crystallographic defects where a nitrogen atom replaces a carbon atom adjacent to a vacant lattice site. This configuration creates a quantum system with unique optical characteristics, including strong photoluminescence (PL) under excitation by green light and the ability to emit fluorescence in the red to near-infrared (NIR) spectral range.

[0062] Features and properties of fluorescent nanodiamond particles include: (i) Biocompatibility: FNDs are generally considered biocompatible due to their carbon-based composition, which is non-toxic and well -tolerated by biological systems, (ii) Photostability: FNDs exhibit excellent photostability, meaning they can emit fluorescence over extended periods without significant degradation, even under continuous illumination, (iii) Brightness and Contrast: The fluorescence emitted by FNDs is bright and stable, providing high contrast imaging capabilities. This makes them suitable for applications where sensitive and precise detection of biological structures or processes is required, (iv) Surface Functionalization: The surface of FNDs can be easily functionalized with various biomolecules, polymers, or targeting ligands. This functionalization allows for specific targeting of FNDs to biological targets, such as cells or tissues, enhancing their utility in targeted drug delivery, bioimaging, and diagnostic applications, (v) Applications: FNDs have been utilized in a wide range of biomedical applications, including but not limited to: (a) Bioimaging: FNDs can be used as contrast agentsFJ ref. UTRGV-P0032WO / Client ref. 2024-0009 for high-resolution imaging of cellular structures and processes due to their bright and stable fluorescence, (b) Drug Delivery: Functionalized FNDs can serve as carriers for therapeutic molecules, enabling targeted delivery to specific cells or tissues, (c) Sensing and Detection: FNDs are employed in biosensors and diagnostic assays for their sensitivity and specific binding capabilities.

[0063] Microglia are the resident immune cells of the central nervous system. Targeting these cells can be particularly important for understanding and treating neurodegenerative disorders, neuroinflammation, and other CNS pathologies. Various moieties (molecules or molecular fragments) can be used to selectively target microglia including but not limited to antibodies and antibody-derivatives such as monoclonal antibodies (mAb) that specifically bind microglia, e.g., mAb to surface markers such as CDl lb, CD45, or P2Y12; ligands and receptor agonists / antagonists such as molecules that bind to receptors predominantly expressed on microglia, such as the purinergic receptors (e.g., P2Y12), toll-like receptors (TLRs), or other immune-related receptors; cytokines and chemokines such as CCL2 (MCP-1) or LL-1 that can interact with receptors on microglia; or peptides and peptide-based agents such as short amino acid sequences or polypeptides that can specifically bind to microglial receptors or antigens.

[0064] TMEM119 (Transmembrane Protein 119) is a transmembrane protein predominantly expressed in microglia, the resident immune cells of the central nervous system (CNS). Identified as a marker specific to microglia, TMEM119 has garnered interest in biomedical research for its potential as a targeting ligand in therapeutic interventions targeting CNS diseases.

[0065] Transmembrane protein 119 precursor (SEQ ID NO: 1) has a signal peptide at amino acids 1 to 25 with amino acids 26 to 283 defining the mature polypeptide. TMEM119 having a transmembrane region from amino acids 97 to 117. NP_859075.2 transmembrane protein 119 precursor [Homo sapiens] MVSAAAPSLLILLLLLLGSVPATDARSVPLKATF LEDVAGSGEAEGSSASSPSLPPPWTPALSPTSMGPQPITLGGPSPPTNFLDGIVDFFRQYV MLIAVVGSL AFLLMFIVC AAVITRQKQKAS AYYP S SFPKKKYVDQ SDRAGGPRAF SEVP DRAPD SRPEEALD S SRQLQ ADIL AATQNLKSPTRAALGGGDGARMVEGRGAEEEEKGS QEGDQEVQGHGVPVETPEAQEEPCSGVLEGAVVAGEGQGELEGSLLLAQEAQGPVGPP ESPCACSSVHPSV (SEQ ID NO: 1).

[0066] Tmeml l9 is not well characterized in humans; however, it is a highly specific cellsurface marker for microglia and is not expressed in circulating monocytes and other immune orFJ ref. UTRGV-P0032WO / Client ref. 2024-0009 neuronal cell types. It has also been tested that blood-derived macrophages also do not express Tmem. This observation makes it a perfect target for M2ND-based drug delivery. Therefore, an investigation into the capacity of Tmeml l9 targeted nano-drug to reach the target site of the inflammation in the brain and deliver anti-HIV drugs on-site was conducted. It is understood that most of the inflammation sites will also have a higher population of HIV-infected microglial cells as a natural inflammatory response. Bringing the drug to that site directly where active virus and virus-infected cells are present will help to control viral replication to a much greater extent than the non-targeted approach. It is worth acknowledging that expression of Tmeml l9 on microglia has shown to be downregulated during several neuronal disorders such as Traumatic Brain Injury, ischemic stroke, Alzheimer's Disease, etc. However, expression of Tmeml l9 expression in the presence of HIV is not well investigated. Observations on HMC3 cells exposed to HIV-1 for 5 days indicated that HIV significantly induced the Tmeml l9 gene (3 fold) and protein (2 fold) expression compared to the control. In addition, exposure to cART drugs (DTG - 20uM, TNF - 1.8uM, and FTC -21uM) with HIV also showed a significant increase in the expression. Volumetric analysis of three different experiments has also established the consistent expression of Tmeml l9 in the microglia cells indicating the importance of this receptor protein as a drug target. Thus, this observation provided strong evidence of the upregulation of Tmeml l9 expression during HIV infection and cART -treated conditions providing for Tmeml 19 target-specific drug delivery.

[0067] PDGF beta, or Platelet-Derived Growth Factor beta, refers to one of the isoforms of the Platelet-Derived Growth Factor. The PDGF family of growth factors play significant roles in cell growth, proliferation, and survival, particularly in cells of mesenchymal origin like fibroblasts, smooth muscle cells, and glial cells. PDGF exists in several isoforms, primarily PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC, and PDGF-DD. These isoforms are dimeric proteins, composed of two chains (A, B, C, or D). PDGF beta or PDGFP (PDGF-BB) refers to the isoform where both chains are of the B type (BB)(one example of PDGFP has an amino acid sequence ofMRLPGAMPALALKGELLLLSLLLLLEPQISQGLVVTPPGPELVLNVS STFVLTC SGS APV VWERMSQEPPQEMAKAQDGTFSSVLTLTNLTGLDTGEYFCTHNDSRGLETDERKRLYIF VPDPTVGFLPNDAEELFIFLTEITEITIPCRVTDPQLVVTLHEKKGDVALPVPYDHQRGFS GIFEDRS YICKTTIGDREVD SD AYYVYRLQ VS SINVS VNAVQT VVRQGENITLMCIVIGNFJ ref. UTRGV-P0032WO / Client ref. 2024-0009EVVNFEWTYPRKESGRLVEPVTDFLLDMPYHIRSILHIPSAELEDSGTYTCNVTESVNDH QDEKAINITVVESGYVRLLGEVGTLQFAELHRSRTLQVVFEAYPPPTVLWFKDNRTLGD SSAGEIALSTRNVSETRYVSELTLVRVKVAEAGHYTMRAFHEDAEVQLSFQLQINVPVR VLELSESHPDSGEQTVRCRGRGMPQPNIIWSACRDLKRCPRELPPTLLGNSSEEESQLET NVTYWEEEQEFEVVSTLRLQHVDRPLSVRCTLRNAVGQDTQEVIVVPHSLPFKVVVISAI LALVVLTIISLIILIMLWQKKPRYEIRWKVIESVSSDGHEYIYVDPMQLPYDSTWELPRDQ LVLGRTLGSGAFGQVVEATAHGLSHSQATMKVAVKMLKSTARSSEKQALMSELKIMS HLGPHLNVVNLLGACTKGGPIYIITEYCRYGDLVDYLHRNKHTFLQHHSDKRRPPSAEL YSNALPVGLPLPSHVSLTGESDGGYMDMSKDESVDYVPMLDMKGDVKYADIESSNYM APYDNYVPSAPERTCRATLINESPVLSYMDLVGFSYQVANGMEFLASKNCVHRDLAAR NVLICEGKLVKICDFGLARDIMRDSNYISKGSTFLPLKWMAPESIFNSLYTTLSDVWSFGI LLWEIFTLGGTPYPELPMNEQFYNAIKRGYRMAQPAHASDEIYEIMQKCWEEKFEIRPPF SQLVLLLERLLGEGYKKKYQQVDEEFLRSDHPAILRSQARLPGFHGLRSPLDTSSVLYTA VQPNEGDNDY1IPLPDPKPEVADEGPLEGSPSEASSTLNEVNTSSTISCDSPLEPQDEPEPEPQLELQVEPEPELEQLPDSGCPAPRAEAEDSFL (SEQ ID N0:2)).

[0068] Microglia play crucial roles in immune surveillance, neuroinflammation, and synaptic maintenance within the CNS. TMEM119 is highly expressed on the cell surface of microglia, distinguishing them from other glial cells and immune cells within the brain. This specificity makes TMEM119 an attractive candidate for targeted drug delivery systems aimed at treating neurodegenerative diseases, brain tumors, and other CNS disorders.

[0069] In the context of nanomedicine, TMEM119 can be utilized as a targeting ligand conjugated to nanocarriers, such as nanoparticles or liposomes. By functionalizing these carriers with TMEM119, researchers can enhance their ability to selectively bind to and penetrate microglia-rich regions of the brain, thereby improving the delivery efficiency of therapeutic agents. This targeted approach not only minimizes off-target effects but also enhances therapeutic efficacy by ensuring that drugs reach their intended cellular targets.

[0070] Furthermore, the specific expression of TMEM119 in microglia provides an opportunity for non-invasive imaging and diagnostic purposes. Fluorescently labeled TMEM119 can be employed in bioimaging techniques to visualize microglia distribution, activation states, and responses to therapeutic interventions in real-time, offering valuable insights into disease progression and treatment outcomes.FJ ref. UTRGV-P0032WO / Client ref. 2024-0009

[0071] However, challenges remain in optimizing the design and delivery of TMEM119- targeted nanocarriers, including ensuring stability, biocompatibility, and sufficient payload capacity for therapeutic molecules. Additionally, further research is needed to fully characterize TMEM1 19's role in neuroimmune responses and its potential implications for CNS therapies.

[0072] In summary, TMEM119 represents a promising targeting ligand in nanomedicine for its specificity to microglia and potential applications in treating CNS disorders. Continued research and development efforts are crucial to harnessing TMEM119's full potential and advancing targeted therapies that can effectively address the complex challenges of neurological diseases.

[0073] Embodiments provide materials and methods for treating and / or diagnosing diseases affecting the central nervous system (CNS) and / or other viral reservoir organs by utilizing nanodiamond (ND) particles conjugated to therapeutic and / or diagnostic agents, such as cART, and microglia targeting agent TMEM119 (M2ND). The surface electrostatic potential of ND makes it possible to attract water molecules and efficiently absorb drug molecules. Thus, M2ND allows the delivery of anti -HIV drugs that are not stable or dispersible in water. Due to ND’s reversible surface electrostatic potential, M2ND exhibits a sustained release of the therapeutic and / or diagnostic agents.

[0074] In addition, M2ND possesses remarkable fluorescent properties and photostability and thereby can be used as an optical probe in the CNS. It can be instrumental as a multifunctional, CNS-trackable delivery vehicle in a single nanostructure. From the site of injection, this ND-cART-Tmeml 19 will be taken up by the circulating monocytes entering the brain and crossing the BBB in response to neuroinflammation. On the brain site, it will be released from the monocytes and selectively attached to microglia with a specific Tmeml l9 surface receptor.

[0075] One aspect of the invention relates to a method comprising administering a therapeutically effective amount of nanodiamonds (NDs) conjugated to anti-retroviral compounds and microglia targeting agent TMEM119, wherein the conjugation administration improves the delivery of anti-retroviral compounds into brain cells and the trackability of the drug delivery due to the fluorescent property of NDs in the subject compared to an administration of the free anti-retroviral compounds at a same dose.FJ ref. UTRGV-P0032WO / Client ref. 2024-0009

[0076] In some embodiments, the composition comprises nanodiamond (ND) particles that are chemically synthesized carbon nanoparticles that feature a diamond core. In certain embodiments, the surface of the ND particles comprises diamond or diamond-like carbons such as, for example, graphite, fullerene (Ceo), layered shells, and / or amorphous carbon. ND particles can be formed using several techniques such as, for example, pulsed laser ablation (PLA), shock wave synthesis, chemical vapor deposition (CVD), and the like. Commercially available ND particles are typically produced by impact events such as an explosion occurring over an extremely short period of time under high pressure and temperature conditions, e.g., over 20 GPa and 3000° C.II. Anti-HIV Therapeutics

[0077] Anti-HIV therapeutics include Nucleoside Reverse Transcriptase Inhibitors (NRTIs), such as Lamivudina (LMV), Emtricitabina (FTC), Zidovudina (ZDV), Tenofovir (TDF), or Abacavir (ABC); Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) such as Nevirapine, Efavirenz, Etravirine, or Rilpivirine; Protease Inhibitors such as Amprenavir, Atazanavir, Darunavir, Fosamprenavir, Indinavir, Lopinavir, Nelfinavir, Ritonavir, Saquinavir, or Tipranavir; Integrase Strand Transfer Inhibitors (INSTIs) such as Raltegravir, Elvitegravir, Dolutegravir (DTG), or Bictegravir; Fusion Inhibitors such as Enfuvirtide; CCR5 Antagonists such as Maraviroc; Capsid Inhibitors such as Lenacapavir (Sunlenca); and Post-Attachment Inhibitors such as Ibalizumab. Combination therapies using combinations of these drugs can be used enhance efficacy or reduce resistance.

[0078] M2ND formulations are developed with self-tracking capabilities. Design of the nanoformulation is required to combine multiple functions without losing the biological activity of the drug molecule that is efficacious, stable, safe, and non-toxic. A complete set of nanoformulations of M2ND with clinically important anti-HIV drugs DTG, TNF and FTC is designed. Therefore, both chemical characterization of an initial M2ND and in vitro uptake and release will be conducted as follows: (A) The M2ND-bio hybrids will be fabricated, and evaluated for their stability, fluorescence profiles, and toxicities. (B) The characterized M2ND - Tmeml l9 antibody conjugates will be evaluated for target-specific tracking efficiencies. The inventor hypothesizes that M2ND will be stable in vitro and will provide consistent fluorescence while tracking microglial cells.FJ ref. UTRGV-P0032WO / Client ref. 2024-0009

[0079] M2ND optimization, and visualization: Using M2ND for drug delivery requires adjustment of the physical properties of the surfaces of particles as well as their characterization (e.g. optical and magneto-optical properties). M2ND surface will be adjusted via chemical modifications and linking to biomolecules for targeting of microglial cells. Therefore, it will be detectable using the ODMR technique using a custom-built microscope. To enhance their drug loading, preserve optical properties, as well as to prevent unwanted interaction with serum proteins, these oxygen-centered surface groups can be terminated using organic capping ligands or / and the introduction of PEG sequences.

[0080] M2ND formulation Preparation: One example of preparation of a M2ND formulation, a well-characterized M2ND can be taken from different batches of M2ND powder and can be characterized using a previously published protocol. With approximately 39,270 carboxyl groups per nanodiamond and assuming one molecule binds to one carboxyl group, it is possible for 39,270 antibody molecules bind with one M2ND particle. To evaluate loading uniformity at the final step, techniques such as Dynamic Light Scattering (DLS), UV-Vis spectroscopy, Raman spectroscopy, and electron microscopy can be employed.

[0081] Transmission Electron Microscopy (TEM) and Raman spectroscopy characterization to observe the formulation size: The preliminary characterization (particle size, shape, and dispersity, etc.) of the M2ND formulation will be conducted using TEM with Energy Dispersive Spectroscopy (EDS) housed at CNM, ANL.

[0082] X-ray Diffraction (XRD) study to understand the shape of the formulation: The ANL has Bruker GADD / D8 X-Ray system with Apex Smart CCD and imaging plate detectors and direct-drive rotating anode.

[0083] The sustained drug release study of M2ND formulation: The initial dissolution of the nanoformulation in a buffer can be measured and subsequent diffusion of the drug in media as per a published protocol. The final drug concentration of the M2ND formulation will be determined using reverse-phase high-performance liquid chromatography (RP-HPLC).

[0084] Selection of M2ND formulation for in vitro study: The selection of the optimized formulation is based on size, PDI, zeta potential, drug loading capacity, and sustained release studies. The formulation with all three cART drugs with Tmeml l9 is given preference considering the translational values of that formulation.FJ ref. UTRGV-P0032WO / Client ref. 2024-0009

[0085] Detection capability of M2ND formulations in the microglia (in vitro) can be assessed. To achieve effective drug delivery, the M2ND formulation should reach the cells in deep brain tissue crossing through BBB which acts as a significant barrier for the many cART drugs. It is contemplated that the M2ND formulations will effectively bind with microglia which can be measured through fluorescence intensity and will effectively control the viral replication within microglia through the sustained release of the cART drugs.

[0086] In vitro uptake and release kinetics study with M2ND in the human PBMC, macrophages, and microglia: This experiment is designed to monitor the uptake and release

[0087] In vitro ADA assay to evaluate the immune response to M2ND formulation: Infliximab Total Antibody ELISA Kit (Cat # ABIN2862668, Antibodies Online, PA).

[0088] Effect of M2ND on primary human neurons and HPM (Inflammatory response): ROS assay is performed to observe any M2ND-induced neuroinflammation as per previous publication and preliminary data.

[0089] Target-specific binding of M2ND formulations with HPM cells: In brief, HPM cells will be seeded in the six-well coverslip containing slides and allowed to adhere. [...] In addition, other brain cells such as primary human astrocytes, brain endothelial cells, glial cells, and neurons (Sciencells, USA) will be tested for M2ND formulation uptake to rule out any indiscriminate uptake of the formulation.

[0090] Migration of M2ND formulations across an artificial BBB: Evaluation of trans- endothelial electrical resistance (TEER) (EVOM voltmeter, World Precision Instrument, USA) and permeability of FITC-labeled dextran (Molecular Probes, USA) can serve as quality controls for BBB “tightness”.

[0091] Antiretroviral efficacy of M2ND in primary human microglia cells: HIV replication will be monitored in infected cell supernatant with p24 ELISA assay (ZeptoMetrix, USA) at 1, 5, 10, and 14 days after drug treatment. The Intact Proviral DNA Assay (IPDA) can be performed in HIV-infected and M2ND-treated cells with digital PCR (dPCR) on a Qiagen QIAcuity One 5Plex digital PCR instrument using the QIAcuity Probe PCR kit.

[0092] Selection of optimized M2ND formulations (Go-No Go decision): Based on the score, the highest 2-3 % for the formulation will be given a Go decision.

[0093] Safety, tracking, and antiretroviral efficacy of M2ND formulations in mouse models. To establish M2ND as a targeted drug delivery tool to the microglia, it is important to considerFJ ref. UTRGV-P0032WO / Client ref. 2024-0009 the amount of drug available for uptake, avoidance of the first-pass effect, and targeted delivery. We hypothesize that the M2ND formulation will be effective, and safe and will provide a trackable delivery of cART drugs to the microglia.

[0094] Pharmacokinetics (PK) evaluation of M2ND formulations: Initial dosing of M2ND in mice can be decided based on the clinical dose of DTG / TNF / FTC (2-30 mg / kg) and interspecies allometric scaling calculation as mentioned in our previous publication. The AUC value will be determined with non-compartmental methods with the log-linear trapezoidal method using Phoenix® WinNonlin (Certara USA, Inc, USA).

[0095] The assessment of M2ND treatment mediated locomotion and cognition changes in mice: BORIS and AnyMaze software will be used to quantify (social, general locomotor, and cognitive performance / leaming) behaviors over time.

[0096] Biodistribution and concentration-specific delivery of M2ND formulation in mice: It is anticipated that a successful nanodrug will clear the circulatory system rapidly within several hours, attain a stable biodistribution within the brain and a significant fraction will be retained within 7 days and up to a month.

[0097] Brain region specific delivery of M2ND formulations (Neuropathological analysis): Mouse microglia and neurons will be identified with biotinylated Griffonia simplicifolia Lectin Llsolectin B4 (Vector Laboratories, Burlingame CA) and neurofilament (Dako) respectively.

[0098] Long-term safety of M2ND formulations: Since M2ND is biocompatible and has shown biodegradation in in vivo, it is expected that the M2ND formulation may not show any systemic toxicity.

[0099] Targeted delivery and tracking of selected M2ND formulations: Inducing neuroinflammation will help the circulating monocytes (M2ND loaded) to reach the brain which will be mimicking the clinical scenario of HIV infection in the brain.

[0100] Therapeutic efficacy of M2ND formulations in the HIV-infected Humanized CD34+ mouse model: A mouse model is selected because of its extended longevity, more robust human immune response, lower rate of graft-vs-host disease, and full HIV infection pathologies with inflammation compared to the limitation of other humanized mouse models.

[0101] Effect of M2ND formulations in reducing the HIV reservoirs in the brain: For HIV-1 LTR gene PCR, and IPDA assay of the brain regions will be performed to observe the viral reservoirs reduction.FJ ref. UTRGV-P0032WO / Client ref. 2024-0009

[0102] Neuronal safety analysis of M2ND formulation through Western Blot: Thus, glutathione synthetase (GSS) level is monitored in the brain considering glutathione as a marker for neuronal injury.III. Examples

[0103] The following examples as well as the figures are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples or figures represent techniques discovered by the inventors to function well in the practice of the invention and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.EXAMPLE I - M2ND STRUCTURE

[0104] The M2ND is reasonably small (50-100 nm or less) with fluorescence capability. The molecular design also includes clinically approved anti-HIV drugs and a unique microglial marker Tmeml l9 on the surface. The M2ND particles can pass through the blood brain barrier of a subject. Due to the electrostatic potential presented by the surface functionalities and the large surface area with respect to ND particles, they facilitate the adsorption of various functional groups and / or drug molecules to a greater extent when compared to other drug carriers. The ND particles can be modified with at least one functional group selected from carboxyls, lactones, ketones, ethers, hydroxyls, and amines.EXAMPLE 2 - TRACKABILITY OF M2ND

[0105] FIG. 1 shows the tracking property of M2ND. To obtain M2ND characterization data, 50, 70, and 100 nm M2ND with carboxylic and hydroxylic surface functional groups were purchased from Adamas Nanotechnologies and subsequently fabricated. To determine the surface structure of the M2ND, Raman spectroscopy, X-ray diffraction (XRD), photoluminescence spectroscopy (PL spectra), and ultra-fast transmission electron microscopy (UEM) analyses were performed. The ND XRD was characteristic of good quality ND having two broad peaks diffracted from atomic planes with Miller indices (111) and (220) of the cubicFJ ref. UTRGV-P0032WO / Client ref. 2024-0009 diamond structure (FIG. 1A - IB). The three different types of red-colored M2ND with characteristic optical properties (peak excitation 570 nm, peak emission ~680nm), as shown in black (Carboxylated, 50 nm), blue (Carboxylated, 70nm), and red (Hydroxylated, lOOnm) spectra. Thus, the tracking potential of M2ND was confirmed using PL measurements (laser excitation 532 nm). These optical properties are due to the presence of NV centers, as observed by UEM. The hydroxylic surface group can be converted into amino function, and both amino and carboxylated ND can be further utilized for surface carbodiimide and click chemistries. The presence of these functional groups allows for the surface chemo- and / and physisorption of different drug and antibody molecules.EXAMPLE 3 - CYTOTOXICITY OF M2ND

[0106] FIG. 2 shows the cytotoxicity evaluation of M2ND on human microglial cells (HMC3). M2ND was exposed at different concentrations (0.01 and O.lmg / ml) to the HMC3 cells to see the direct effect on microglia considering that being the target cells in the brain. Experimentally, HMC3 cells were treated with M2ND formulation for 24 hours, and then, the MTT assay was performed in triplicates (six replicates each time) as per published protocol. HMC3 cells that were not treated with any M2ND were considered as negative control. There was no significant reduction in cell viability up to 0.1 mg / ml. Based on the optimization of the drug loading capacity of M2ND (data not shown) and cytotoxicity, O. lmg / ml of 50 NM M2ND was used for further in vitro and in vivo study (FIG. 2). The long-term toxicity will be observed in in vitro and in vivo studies.

[0107] Safety evaluation of M2ND on Primary Human Microglia Cells (HPM): To observe the safety of M2ND on microglia cells, MTT assays were performed on HPM. Briefly, M2ND was exposed at different concentrations (0.01 - Img / ml) to the HPM cells to see the direct effect on microglia considering that being the target cells of M2ND. This range of concentration is considered based on previous publications. Experimentally, HPM cells were treated with M2ND formulation for 24 hours, and then, the MTT assay was performed in triplicates (six replicates each time) as per published protocol. The untreated HPM cells were considered as negative control. There was no significant reduction in cell viability up to 0.1 mg / ml. Based on the optimization of the drug loading capacity of M2ND (data not shown) and cytotoxicity, O.lmg / ml of 50 nm M2ND was used for further in vitro and in vivo studyFJ ref. UTRGV-P0032WO / Client ref. 2024-0009EXAMPLE 4 - OXIDATIVE STRESS INDUCED BY M2ND

[0108] It was important to evaluate whether M2ND induces any oxidative stress to the targeted microglia cells which are associated with neuroinflammation. M2ND-induced oxidative stress evaluation through reactive oxygen species (ROS) production was conducted. FIG. 3 shows the intracellular ROS production in HMC-3 cells exposed to different concentrations of M2NDs. HMC3 cells were treated with 0.01, 0.1, and Img / ml of all three M2ND for 24hrs and ROS assay was performed. Briefly, cells were cultured in 96-well black clear-bottomed plates. Then untreated (control) and M2ND-treated cells were exposed to dichlorofluorescein diacetate in each well and incubated for 1 h (Molecular Probes, USA). Cells with no drug (untreated) were used as a control and the cells were treated with H2O2 for positive controls (three independent experiments with six replicates each). After 2 h, the first reading of ROS production was read in the BioTek Synergy HT microplate reader (excitation, 485 nm; emission, 528 nm) and ROS production increased with increasing concentration of all M2NDs. A graphical representation was made with RFU vs. treatment with one-way ANOVA analysis (FIG. 3).

[0109] The evaluation of M2ND-induced oxidative stress (Reactive oxygen species (ROS)): It was important to evaluate whether M2ND induces any oxidative stress to the targeted microglia cells that are associated with neuroinflammation. Thus, HPM cells were treated with 0.01, 0.1, and Img / ml of all three M2ND for 24hrs and ROS assay was performed. The ROS production was increased with increasing concentrations of 70 and 100 M2NDs. Whereas 50nm M2ND did not change ROS production indicating the compatibility of this particle for drug development.EXAMPLE 5 - STABILITY OF M2ND IN ROOM TE PERATURE

[0110] A formulation with M2ND at the core and with surface modification with COOH- (50 and 70 nm), and OH- (lOOnm) groups is fabricated. It was conjugated with TNF, FTC, and DTG. Adsorption of drugs on the M2ND surface was performed by adding ND suspension to the DMSO-water (1:9) solution of drugs (2.5 mg / mL). All three formulations appeared clear and colorless by general observation as indicated in FIG. 4. The physical characterization of all the formulations indicated that they can be in solid and powder form in dried condition, stable at room temperature, at -20C for long-term storage. The liquid formulation is stable at pH 7.2-7.5 which provides a physically stable formulation.FJ ref. UTRGV-P0032WO / Client ref. 2024-0009

[0111] As shown in Table 1, both the XRD and Raman study showed no graphitization of - COOH, -NH2 modified ND in all the properties. The measured XRD pattern (Mo Ka-radiation) of nanodiamond powder was a characteristic of good-quality nanodiamonds. Two broad peaks correspond to diffraction from atomic planes with Miller indices (111) and (220) of the cubic diamond structure. Raman spectra were collected using Ar+ ion laser excitation (X = 514.5 nm) and showed vibrational features at 1326 cm1which were a first-order diamond peak. The peak at 1619 cm1was a superposition of sp2 carbon (G-band) and OH groups on the surface.Table 1. Three M2ND (50, 70, and 100 nM) physical and chemical characterizations.EXAMPLE 6 - M2ND PREPARATION, OPTIMIZATION, AND VISUALIZATION

[0112] Using M2ND for drug delivery will require adjustment of the physical properties of the surfaces of particles as well as their characterization (e.g., optical and magneto optical properties). The properties of M2ND, carbon-based materials, obtained synthetically by the high- energy processing of graphite have novel properties at the nanoscale and the high surface area- to-volume ratio. Moreover, M2ND possesses remarkable fluorescent properties and photostability. These ND centers can be initialized, manipulated, and read optically under ambient conditions, and can be used as probes to detect and visualize biological processes within a single cell or even a single molecule. M2ND surface will be adjusted via chemical modifications and linking to biomolecules for consequent targeting of microglial cells in the brain. Therefore, it will be detectable using the ODMR technique using a custom-built microscope. ODMR is a double resonance technique by which the electron spin state of a crystalFJ ref. UTRGV-P0032WO / Client ref. 2024-0009 defect may be optically pumped for spin initialization and readout. Therefore, ND size distribution and colloidal stability can be improved before using them in nano formulation with a drug. This will reduce unwanted interactions with biomolecules while improving controllable drug loading capacity. To enhance their drug loading, preserve optical properties, as well as to prevent unwanted interaction with serum proteins, these oxygen-centered surface groups will be terminated using organic capping ligands and / or the introduction of PEG sequences.

[0113] To prepare the M2ND formulation, a well-characterized M2ND will be taken from different batches of M2ND powder. Those M2ND powders can be mixed with pure DTG, TNF and FTC (Sigma-Aldrich) in DMSO, and the mixture will be sonicated for 5min at room temperature and incubated on magnetic agitation for 12 hours to facilitate the drug adsorption on the M2ND surface. After incubation, the M2ND and solvent will be separated by centrifugation and dialysis to get rid of any extra unabsorbed drugs. Further, the formulation will be characterized by UEM, XRD, and Raman spectroscopy to confirm the molecular conjugation of the drug and M2ND assembly. The M2ND formulation can also be characterized based on its size and surface charge. The final drug concentration of the formulation can be measured through HPLC. Finally, the M2ND formulation can be bio-conjugated with a Tmeml l9 at the concentration of 1 :200 for target-specific delivery. The surface carboxyl group can be directly linked to the amino group of the antibody molecule via carbodiimide coupling using Sulfo- NHS / EDC reagents. On the other hand, hydroxylated M2ND will be used as a starting material to obtain an amino-functionalized surface through pretreatment with (3- aminopropyljtrimethoxysilane (APS) linker.EXAMPLE 7 - DRUG LOADING CAPACITY OF M2ND FORMULATION

[0114] FIG. 5 shows the natural fluorescence of the M2ND while absorbed inside the cells. For the study, cells were grown in vitro and then exposed to the M2ND formulation. After 8 hours of incubation, the drug-loaded cells were observed under the fluorescent microscope and compared with untreated cells. This shows the tracking capability of this formulation within the biological system without adding any additional trackers.EX AMPLE 8 - CELLULAR UPTAKE OF M2ND FORMULATIONFJ ref. UTRGV-P0032WO / Client ref. 2024-0009

[0115] FIG. 6 shows the capability of M2ND taken by microglia cells. In the present study, an M2ND-based nanodrug containing a cART drug, dolutegravir (DTG), was used. HMC3 cells (an immortalized human microglial cell line) were exposed to M2ND-DTG formulation (5mg / ml DTG initial concentration) for 8hrs and at each time point (0, 2, 4, 6, and 8hrs) cells were harvested, the drug was extracted from cell pellet and measured by LC-MS / MS compared with free drug DTG (DTG). Results were expressed as the total DTG accumulated in the cells compared to the initial concentration (FIG. 6). Compared to the DTG, the M2ND-DTG formulation showed a sustained increase in cellular uptake within 4 hours and showed a significant increase up to 8 hours (FIG. 6). Overall, M2ND-DTG formulation showed a sustained increase in drug loading whereas the free DTG retention decreased with 6 hours.

[0116] In vitro human peripheral blood mononuclear cells (PBMC) cellular uptake of M2ND-DTG / FTC formulations: It was important to investigate whether the M2ND formulations can be taken up by the human PBMC when exposed in vitro and in vivo. This data will provide evidence of whether PBMC can be an effective carrier of the M2ND formulations from the site of administration and carry it toward the target site. Overall, M2ND-DTG / FTC formulation showed a sustained increase in drug loading whereas the free DTG and FTC retention did not increase with time. This observation provided evidence that PBMC can be a stable carrier for the M2ND formulations that can be further expanded with other formulations.EXAMPLE 9 - TRANSMEMBRANE PROTEIN 1 19 (TMEMI 1 ) AS TARGET D DRUG DELIVERY

[0117] Tmeml 19 is a highly conserved transmembrane protein very specific for microglia. Its function is not well characterized in humans; however, it is a highly specific cell-surface marker for microglia and is not expressed in circulating monocytes and other immune or neuronal cell types. It has also been tested that blood-derived macrophages also do not express Tmem. This observation makes it a perfect target for M2ND-based drug delivery. Therefore, an investigation into the capacity of Tmeml 19 targeted nano-drug to reach the target site of the inflammation in the brain and deliver anti-HIV drugs on-site was conducted. It is understood that most of the inflammation sites will also have a higher population of HIV-infected microglial cells as a natural inflammatory response. Bringing the drug to that site directly where active virus and virus-infected cells are present will help to control viral replication to a much greater extent than the non-targeted approach. It is worth acknowledging that expression of Tmeml 19 onFJ ref. UTRGV-P0032WO / Client ref. 2024-0009 microglia has shown to be downregulated during several neuronal disorders such as Traumatic Brain Injury, ischemic stroke, Alzheimer's Disease, etc. However, expression of Tmeml l9 expression in the presence of HIV is not well investigated. Observations in HMC3 cells exposed to HIV-1 for 5 days indicated that HIV significantly induced the Tmeml l9 gene (3 fold) and protein (2 fold) expression in HMC3 cells compared to the control. In addition, as shown in FIG. 7A and FIG. 7B, exposure to cART drugs (DTG - 20uM, TNF - 1.8uM, and FTC -21uM) along with HIV also showed a significant increase in the expression. As shown in FIG. 7C, the volumetric analysis of three different experiments has also established the consistent expression of Tmeml l9 in the microglia cells indicating the importance of this receptor protein as a drug target.

[0118] Preliminary data also indicated that Tmeml 19 is not expressed in human PBMC, but is expressed in HMC3, HPM, and also in mouse microglia (SIM-A9) cells. This observation provides strong evidence that circulating PBMC will not bind with the formulations. It will rather carry it inside the cells protecting from other non-specific binding to other immune cells and deliver to the brain inflammation site where a large amount of HIV-infected microglia are present with higher expression of Tmeml 19 protein.EXAMPLE 10 - ANTIRETROVIRAL EFFICACY STUDY OF M2ND FORMULATIONS OF TNF, DTG, AND FTC

[0119] FIG. 8 shows the antiretroviral efficacy of M2ND formulations of dolutegravir (DTG), tenofovir (TNF), and emtricitabine (FTC) compared to their respective free drugs (FD). PWHHMC3 cells were infected with HIV-1 JR-FL for 24 hours and then treated separately with M2ND formulations (ND-TNF (1.8 pM), ND-DTG (20 pM) & ND-FTC (21 pM)) or Free drugs of TNF, FTC, and DTG respectively. These concentrations were selected based on clinically relevant concentrations of the drugs. The culture supernatant was collected after 5 days of incubation to perform an ELISA assay (FIG. 8). This data strongly supported the fact that M2ND will be significantly potent in controlling the virus with the treatment of a single dose. Based on these observations, M2ND will likely be therapeutically effective in reducing HIV infection in human microglia.FJ ref. UTRGV-P0032WO / Client ref. 2024-0009EXAMPLE 11 - SAFETY PROFILE OF THE M2ND FORMULATIONS IN MICE

[0120] Table 2 shows the enzyme levels related to liver or renal toxicity after treatment of M2ND. To observe the safety of 50 nm M2ND formulation on systemic toxicity, the toxicity of M2ND in the BALB / cJ mouse was evaluated. In this experiment, an intravenous injection (i.v) of the M2ND into one group of BALB / cJ mice was given to study the immunohistochemistry and systemic toxicity. To investigate systemic toxicity, mice were observed for 7 days after injection and sacrificed for immunohistochemical and serum chemistry analysis, respectively. The data was compared with control mice. For histopathological analysis, tissue sections were stained with hematoxylin and eosin (H&E stain). Histopathological evaluations were performed as per the guidelines of the Society of Toxicologic Pathology. The pathological analysis did not indicate any neurotoxicity with the M2ND. Serum chemistry of liver and renal profiling was conducted in treated mice by collecting the plasma samples. The overall data showed no changes in enzyme levels related to liver or renal toxicity as explained in Table 2. Thus, it is likely that the M2ND formulation will be safe since cART drugs are clinically approved and M2ND itself is nontoxic to mice.Table 2 shows the safety profile of the M2ND formulations in mice.EXAMPLE 12 - ANTIRETROVIRAL EFFICACY OF M2ND NANO-FORMULATED (ND) TNF, DTG, AND FTC COMPA ED TO THEIR RESPEC TIVE FD

[0121] A study was conducted to observe the antiretroviral efficacy of M2ND formulations with preoptimize concentrations of the clinically relevant ratio of DTG, FTC & TNF (together called ND-cART) against HIV-1 infection in hu-PBL mice. Experimentally, hu-PBL mice were infected with laboratory-grown HIV-1 virus. One group of mice was treated with one dose of combined ND-cART [ND-TNF (31mg / kg), ND-DTG (2mg / kg) and ND-FTC (1.6mg / kg)] and the other group was treated with the same dose of the combined free drug or FD (cART). TheFJ ref. UTRGV-P0032WO / Client ref. 2024-0009 infected and untreated group was considered a positive control. At the end of 14 days, mice were sacrificed. The brain tissues were used for HIV-LTR gene expression through PCR. The HIV- LTR gene expression study has indicated that combined M2ND (ND-cART) treated brain tissue had significantly reduced HIV-LTR gene expression compared to combined FD-treated mice. The PCR gene expression data was expressed as fold changes compared to control mice (FIG. 9A). This observation establishes the efficacy of the M2ND formulations over FD-cART drugs in vivo. The same brain tissue was tested for Tmeml l9 gene expression through RT-PCR in HIV-infected (control) mice and compared with uninfected and untreated mice (negative control) (FIG. 9B). FIG. 9B confirmed that Tmeml l9 gene expression was significantly upregulated (10- fold) in these mice during HIV infection which is suitable for testing the hypothesis.

[0122] HIV and cART-induced Tmeml l9 expression in a humanized mouse model: Even though the Tmeml l9 expression in microglia varies depending on the wide range of neurological disorders, these studies did not discuss the HIV infection on Tmeml l9 expression in microglia. Preliminary data and previous literature indicating the higher expression of Tmeml 19 even during HIV-infected and cART -treated conditions support the approach.

Claims

FJ ref. UTRGV-P0032WO / Client ref. 2024-0009CLAIMS1. A nanodiamond composition comprising a microglial targeting moiety and an anti-HIV drug operatively coupled to a nanodiamond particle.

2. The composition of claim 1, wherein the microglial targeting moiety is Tmeml 19 or PDGFp.

3. The composition of claim 1, wherein the nanodiamond particle is modified with at least one functional group selected from carboxyls, lactones, ketones, ethers, hydroxyls, and amines.

4. The composition of claim 1, wherein the anti-HIV drug is selected from dolutegravir, tenofovir, emtricitabine, lamivudine, abacavir, zidovudine, efavirenz, rilpivirine, doravirine, atazanavir, darunavir, lopinavir / ritonavir, bictegravir, raltegravir, elvitegravir, enfuvirtide, maraviroc, and lenacapravir.

5. The composition of claim 1, wherein the nanodiamond composition is between 10-100 nm in diameter.

6. The composition of claim 1, wherein the nanodiamond particle is detectable via ELISA, X-ray diffraction, photoluminescence spectroscopy, or ultra-fast transmission electron microscopy.

7. The composition of claim 1, wherein the nanodiamond particle has a reversible surface electrostatic charge.

8. A method of treating HIV, comprising administering a nanodiamond composition comprising a microglial targeting moiety and an anti-HIV drug operatively coupled to a nanodiamond particle.

9. The method of claim 8, wherein the microglial targeting moiety is Tmeml 19 or PDGF0.FJ ref. UTRGV-P0032WO / Client ref. 2024-000910. The method of claim 8, wherein the nanodiamond particle is modified with at least one functional group selected from carboxyls, lactones, ketones, ethers, hydroxyls, and amines.

11. The method of claim 8, wherein the anti -HIV drug is selected from dolutegravir, tenofovir, emtricitabine, lamivudine, abacavir, zidovudine, efavirenz, rilpivirine, doravirine, atazanavir, darunavir, lopinavir / ritonavir, bictegravir, raltegravir, elvitegravir, enfuvirtide, maraviroc, and lenacapravir.

12. The method of claim 8, wherein the nanodiamond composition is between 10-100 nm in diameter.

13. The method of claim 8, wherein the nanodiamond composition is detected via ELISA, X- ray diffraction, photoluminescence spectroscopy, or ultra-fast transmission electron microscopy.

14. The method of claim 8, wherein the nanodiamond particle has a reversible surface electrostatic charge.FJ ref. UTRGV-P0032WO / Client ref. 2024-000915. A method of imaging microglia in a subject, comprising administering to the subject a nanodiamond composition comprising a microglial targeting moiety operatively coupled to a nanodiamond particle, wherein the nanodiamond particle exhibits fluorescence due to nitrogenvacancy (NV) centers; and detecting the nanodiamond composition in the brain of the subject.

16. The method of claim 15, wherein the microglial targeting moiety is Tmeml 19 or PDGF0, and the nanodiamond composition is between 10-100 nm in diameter.

17. A method of treating a central nervous system (CNS) disorder in a subject, comprising administering a therapeutically effective amount of a nanodiamond composition comprising a microglial targeting moiety and a therapeutic agent operatively coupled to a nanodiamond particle, wherein the therapeutic agent is selected from the group consisting of anti-HIV drugs, anti-inflammatory agents, neuroprotective agents, and combinations thereof.

18. The method of claim 17, wherein the CNS disorder is selected from the group consisting of HIV-associated neurocognitive disorders, Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, and traumatic brain injury.

19. A nanodiamond composition comprising:(a) a nanodiamond particle modified with at least one functional group selected from carboxyls, lactones, ketones, ethers, hydroxyls, and amines;(b) a microglial targeting moiety operatively coupled to the nanodiamond particle; and(c) a diagnostic agent operatively coupled to the nanodiamond particle, wherein the diagnostic agent is a fluorescent label or a contrast agent.

20. The composition of claim 19, wherein the microglial targeting moiety is Tmeml 19, and the nanodiamond particle has a reversible surface electrostatic charge to facilitate sustained release of the diagnostic agent.

21. A method of preparing a nanodiamond composition for targeted drug delivery, comprising:FJ ref. UTRGV-P0032WO / Client ref. 2024-0009(a) providing nanodiamond particles having a diameter of 10-100 nm and modified with at least one functional group selected from carboxyls, lactones, ketones, ethers, hydroxyls, and amines;(b) conjugating a microglial targeting moiety to the nanodiamond particles via covalent or non- covalent interactions; and(c) adsorbing an anti-HIV drug onto the nanodiamond particles, wherein the anti-HIV drug is selected from dolutegravir, tenofovir, emtricitabine, or combinations thereof.

22. The method of claim 21, further comprising characterizing the nanodiamond composition using at least one technique selected from dynamic light scattering (DLS), UV-Vis spectroscopy, Raman spectroscopy, or electron microscopy to confirm size, surface charge, or drug loading.

23. A pharmaceutical composition comprising:(a) a plurality of nanodiamond particles, each having a diameter of 10-100 nm and a surface modified with at least one functional group selected from carboxyls, lactones, ketones, ethers, hydroxyls, and amines;(b) a microglial targeting moiety conjugated to the nanodiamond particles; and(c) a therapeutic agent adsorbed onto the nanodiamond particles, wherein the pharmaceutical composition is formulated for intravenous administration and capable of crossing the blood-brain barrier (BBB) to target microglia in the brain.

24. The pharmaceutical composition of claim 23, wherein the therapeutic agent is an anti- HIV drug, and the nanodiamond particles exhibit sustained release of the therapeutic agent in the brain due to reversible surface electrostatic potential.

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