Functionalized lipid nanoparticles for targeted delivery to endometrium

Abstract

LNPs can penetrate uterine mucosa to deliver therapeutic or prophylactic agents such as functional nucleic acids to the endometrium. Targeting of the LNPs using ligands expressed at particular times in an endometrial cycle ensures that the LNPs penetrate and release the mRNA primarily to the tissue associated with the peak time period for implantation, thereby providing a means to address infertility, as well as for treatment of other disorders such as cancer.

Description

[0001]FUNCTIONALIZED LIPID NANOPARTICLES FOR TARGETED DELIVERY TO ENDOMETRIUM CROSS REFERENCE TO RELATED APPLICATION This application claims benefit of and priority to U.S. Provisional Application No. 63 / 687,525, filed August 27, 2024, which is specifically incorporated by reference herein in its entirety. REFERENCE TO THE SEQUENCE LISTING The Sequence Listing submitted as an XML filed named “JHU_C_17814_PCT_ST26.xml”, created on August 19, 2025, and having a size of 8,127 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.834(c)(1). FIELD OF THE INVENTION This invention is generally in the field of tissue-specific targeted drug delivery. FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT This invention was made with government support under Grant Nos. HD103124 and HD108905 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND OF THE INVENTION The female reproductive tract is divided into the upper and lower genital tract. The upper genital tract (UGT) is comprised of the cervix, uterus, fallopian tubes, and ovaries, while the lower genital tract consists of the vagina and vulva. The UGT is a target for various health issues, such as tumors, adhesions, infections, and inflammation. The uterus is a multifaceted reproductive organ, which harbors a complex environment characterized by dynamic changes in microbiome, hormones, immune composition, and other physiological milieu that change throughout the life span of women. The etiology of many diseases that affect the UGT have origins in the local tissues. Therefore, these diseases require targeted delivery of therapeutics to achieve sufficient local drug exposure. However, efficient delivery is constrained by the UGT’s complex anatomy and dynamic physiology. Further, the safety of such systems must be clearly established not only for the women but also for the growing fetus in the case of pregnancy. Female UGT diseases affect a large number of women each year. Some are associated with serious health consequences and high economic burden. As of 2021, globally, 9% of women of reproductive age including 1.5 million women in the United States, are infertile. The burden of infertility in developing countries can reach up to 30% of reproductive-aged 47 JHU C 17814 PCT 095238 / 00572 women. Infertile women are 6–8 times more likely to have endometriosis, a chronic inflammatory disease characterized by the presence of ectopic endometrial tissues. The estimated global prevalence of endometriosis is approximately 10% of all reproductive-age women. The endometrium, the mucosal layer of the uterus, is important for its barrier and reproductive functions1. Dysfunction in the endometrium is characteristic of a myriad of women’s health conditions, including uterine fibroids, adhesions, Asherman’s Syndrome, endometriosis, infections, and cancers2-5. Thinning in the endometrial lining, spontaneous or secondary to a gynecologic condition, is associated with implantation failures and poor pregnancy outcomes6-8. Even with in vitro fertilization (IVF), women with a thin endometrium (TE) are less likely to become pregnant9-13. There are limited evidence-based treatment options for TE14. Cell therapies can potentially regenerate TE15,16, but an effective pharmacologic approach would be more accessible to patients. Few clinical studies attempted intrauterine administration of growth hormones and cytokines to increase uterine blood flow and endometrial thickness before embryo transfer in IVF17-24. However, challenges such as insufficient endometrial targeting, systemic exposure, and the inability to sustain adequate therapeutic protein levels within the uterus hinder both efficacy and safety19. Messenger RNA (mRNA) can induce and sustain local therapeutic protein expression25-27. Messenger RNA (mRNA) is an efficient method to introduce intracellular or secreted proteins into target cells. Delivering mRNA encoding for growth factors, cytokines, or gene editors could provide new therapeutic options for endometrial disorders. Lipid nanoparticles (LNPs) are promising delivery vehicles for mRNA in vivo, but they lack tissue specificity and show wide biodistribution even after local administration. It is therefore an object of the present invention to provide new options for targeted mRNA delivery to the UGT, especially the endometrial tissues. SUMMARY OF THE INVENTION Compositions for targeted endometrial delivery of one or more active agent are disclosed. The compositions include surface receptor-targeted ligand (TL) functionalized lipid nanoparticles (LNPs) (herein, “TL-LNPs”) as the carrier for the active agent. Targeted surface receptors on the endometrium that are over-expressed during the implantation window include integrins, cadherins, selectins, and immunoglobulins. In some forms, the TL-LNPs include four components: (i) an ionizable lipid (IL); (ii) a helper lipid (HL); 2 JHU C 17814 PCT 095238 / 00572 (iii) at least one targeting ligand / moiety functionalized phospholipid (TL-PL), where the at least one targeting ligand / moiety functionalized phospholipid is an integrin targeting ligand (ITL-PL); and (iv) a lipid (for example, phospholipid)-anchored polymer such as polyalkylene glycol for example, polyethylene glycol (PEG-lipid). “PEG-lipid” is used herein generally to refer to the lipid-anchored polymer component of the LNP. The lipid-polymer molecules contain two parts. The polymer part can be PEG, poly(oxazoline), poly(vinyl alcohol), poly(glycerol), poly(N- vinylpyrrolidone), polyaminoacids and poly N-(2- hydroxypropyl)methacrylamide]. The lipid component in the PEG-Lipid can be phosphatidylethanolamine lipid comprising C8, C10, C12, C14, C16, C18, C20, 0-3 unsaturated bonds, dilauroylglycerol, dimyristoylglycerol, dipalmitoylglycerol, distearoylgiycerol, dilaurylglycamide, dimyristylglycamide, dipalmitoylglycamide, disterylglycamide, cholesterol, or saturated and unsaturated fatty acids (C8-C20) such as stearic acid and oleic acid. In some forms, the PEG in the PEG-lipid is a low molecular weight PEG ( preferably from about 200, about 300, 350-550 Daltons). In some forms the PEG in the PEG-lipid is a high molecular PEG (2000-5000 daltons), and the PEG-Lipid is a PEG-sheddable PEG-Lipid. Sheddable is any PEG-lipid that has a lipid acyl chain 14 carbons in length or shorter. Non-shedabble PEG is any PEG-lipid that has a lipid acyl 18 carbons in length or longer. Efficacy has been demonstrated with both sheddable PEG-lipid and non-sheddable PEG-lipid with short PEG (350). The lipid anchor in the PEG-Lipid is preferably a C14-C18 phospholipid. Thus the LNPs can include PEG-dilauroylglycerol, PEG-dimyristoylglycerol (PEG- DMG), PEG-dipalmitoylglycerol, PEG-distearoylgiycerol (PEG-DSPE), PEG- dipalmitoylglycerol, PEG-disterylglycerol, PEG-dilaurylglycamide, PEG- dimyristylglycamide, PEG-dipalmitoylglycamide, PEG-disterylglycamide, PEG-cholesterol, and PEG-DMB (3,4-Ditetradecoxylbenzyl- [omega]-methyl-poly(ethylene glycol) ether), 1,2- dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]), PEG conjugated to ceramides. The density of the targeting ligand (such as an integrin targeting ligand (ITL)) on the surface of the LNP is effective to selectively target the LNP when administered locally, to the endometrium, with negligible trafficking to non-desirable sites such as the liver. In some forms, the ITL-PL is at a concentration from about 0.5 to about 10 mol% of the total lipid concentration in the LNP, for example, between about 1.5. to about 7 mol% of the total lipid 3 JHU C 17814 PCT 095238 / 00572 concentration in the LNP, for example, about 1, 2, 3, 4, 5, 6 or 7 mol% of the total lipid concentration in the LNP. In some forms, the ITL-PL includes a linker between the integrin targeting ligand (ITL) and the phospholipid (PL), for example, a PEG linker. In some forms, no polymer such as PEG linker is present, because there is some sort of linker between the peptide and the PL (such as azideAzide / DBCO or thiol / maleimide linker). The active agent can be, for example, a therapeutic, diagnostic, prophylactic compound, or a combination thereof. The active agent can also include or be a protein, peptide, carbohydrate, polysaccharide, nucleic acid molecule, and / or organic small molecule. In a particular embodiment, the active agent is a nucleic acid such as an mRNA. Methods for targeted endometrial delivery of one or more active agents to a subject in need thereof, are also disclosed. The method incudes administration of one or more active agents using the LNPs targeted to integrins, cadherins, selectins, and immunoglobulins to a subject in need thereof, using a route of administration and timing of administration that is effective to target elevated integrin expression at the site of administration. Accordingly, the method involves spatiotemporal targeting, which entails delivering one or more therapeutics specifically to the endometrium excluding the myometrium, liver, spleen, lung, etc. (spatial), and specifically during the implantation window in the reproductive cycle (temporal). Exemplary subjects include subjects with one or more upper genital tract diseases / infections / conditions, for example, endometriosis, endometrial cancer, a subject in need of infertility treatments, etc. BRIEF DESCRIPTION OF THE DRAWINGS FIGs.1A-1H show the physicochemical characterization and in vivo delivery efficiency of RGD-LNPs. FIG.1A shows the formulation characterization of untargeted LNP (Control-LNP) and LNP functionalized with 5 mol% C18-RGD (RDG-LNP). Both LNPs were co-formulated with 1.5 mol% C18-PEG350 as the PEG-lipid stabilizer component. Data represent the mean ± SEM (n=3). (FIGs.1B-1D) The diffusion of nanoparticles on mouse endometrial surface obtained on day 5 p.c. FIG.1B shows time-resolved changes in MSD of fluorescently labeled PS-COOH and control- and RGD-LNP. Data represent the mean ± SEM of n=8-12 measurements FIG.1C shows distribution histograms of Log10(MSD t = 1 sec). Data represent the mean ± SEM of 3 biological replicates, each replicate representing the average of 8-12 measurements. The highlighted area represents the error bands. The % particle distribution histograms were fitted into a Gaussian model using a non-linear regression of 189 degrees of freedom to calculate the mean Log10(MSD t = 1sec) ± SEM. The dotted red line indicates the fraction of nanoparticles showing rapid diffusion on the 4 JHU C 17814 PCT 095238 / 00572 endometrial surface with Log10(MSD t = 1 sec) > -1 FIG.1D shows quantification of mobile nanoparticle fraction showing Log10(MSD t = 1 sec) > -1. Data represent the mean ± SEM of 3 biological replicates. FIG.1E shows the luciferase expression normalized to mg protein in tissue homogenates 4 hr. post intrauterine infusion of 20 µL containing 2 µg fLuc mRNA into the mid-distal right uterine horn at day 5 p.c in female CD-1 mice. Whole uteruses were collected for luciferase quantification. Data represent the mean ± SEM (n=5 for LNP groups and n=3 for the untreated group). FIG.1F shows luciferase expression normalized to mg protein following intrauterine infusion of 20 µL containing 2 µg fLuc mRNA into the mid- distal right uterine in unmated female CD-1 mice. Data represent the mean ± SEM (n=6). Figure 1G shows the uterus-to-liver and FIG.1H shows the uterus-to-spleen luciferase expression ratios calculated from (FIG.1E) and (FIG.1F). Data represent the mean ± SEM (n=5-6). Statistical significance was calculated using Fisher's LSD test. FIGs.2A-2K show the screening of RGD conjugation strategies enables the discovery of LNP compositions showing high endometrium targeting and low off-target exposure. FIG. 2A is a schematic diagram illustrating the experiment design. A library of LNPs was infused intrauterine in mice at day 5 p.c. to evaluate the uterus targeting efficiency and intravenously in healthy unmated mice to characterize their systemic tropism. FIG.2B and 2G are a library of 12 LNPs with varying degrees of RGD-lipid amounts, the site of RGD conjugation, and the type of PEG-lipid stabilizer component used in LNP formulation. In FIG. 2C-2G, 20 µL containing 2 μg fLuc mRNA-LNPs was infused into the right uterine at day 5 p.c. followed by luciferase expression quantification in the whole uterus, liver, and spleen 4 hr. post- infusion. FIG.2C shows LNPs A-C co-formulated with C18-PEG350 show the effect of varying the amounts of C18-RGD on luciferase expression. Data represent the mean ± SEM (n=4-5). FIG. 2D shows the extent of RGD-mediated uptake in the uterus and comparison between a spacer and spacer-free RGD conjugation methods on luciferase expression. LNPs D and E contain varying 1.5 and 5 mol% C18-PEG2000-RGD, respectively. For the vitronectin inhibition experiment, mice were injected with a 10 µL solution containing 0.5 mg / mL vitronectin 5 min before intrauterine injection of LNP C. Data represent the mean ± SEM (n=4). FIG. 2E shows LNPs F-H co-formulated with C18-PEG2000 show the effect of varying the amounts of C18-RGD on luciferase expression. Data represent the mean ± SEM (n=4). FIG. 2F shows LNPs I-K co-formulated with C14-PEG2000 (DMG-PEG2000) show the effect of varying the amounts of C18-RGD on luciferase expression. Data represent the mean ± SEM (n=4). FIG.2G shows luciferase expression of LNP L containing 5 mol% C18- RGD and co-formulated with C14-PEG350 as compared to LNP C . Data represent the mean 5 JHU C 17814 PCT 095238 / 00572 ± SEM (n=3-4). FIG.2H shows luciferase expression in the liver 4 hr following tail-vein injections of LNPs A-L containing 2 μg fLuc mRNA in unmated healthy female CD-1 mice. Data represent the mean ± SEM (n=3 for all LNPs, except n=6 for LNP C). FIG.2I shows efficiency of uterus targeting of LNPs A-L following intrauterine infusions on day 5 p.c. as quantified by calculating the percentage of luciferase expression originating from the uterus relative to the total expression in the liver, spleen, and uterus. Data represent the mean ± SEM (n=4-5 for all LNPs, except LNP C n=17) FIG.2J shows liver tropism following intravenous injection of LNPs A-L in unmated healthy female mice. Data are shown as fold changes in liver expression relative to that of LNP C. FIG.2K is a schematic diagram proposing a new LNP engineering concept by employing Bi-functional Ligands via Organized Conjugation (BLOC) for maximizing tissue-specific targeting and reducing off-target systemic exposure following local injection. FIGs.3A-3F show the biodistribution and kinetics of GM-CSF expression. FIG. 3A shows baseline levels of mouse GM-CSF in untreated mouse tissues as quantified by ELISA. Data represent the mean ± SEM (n=3). FIG.3B shows GM-CSF levels in mice treated with fLuc mRNA-loaded LNP C. After 4 hr of intrauterine infusion, organs were collected, homogenized, and quantified for GM-CSF level using ELISA. Data represent the mean ± SEM (n=3). FIG.3C shows 20 μL containing 4 μg GM-CSF mRNA in LNP C were injected in the right uterine horn in mice at day 5 p.c. After 4 hr, tissues were harvested, homogenized, and quantified for their GM-CSF levels using ELISA. Data represent the mean ± SEM (n=3). FIGs.3D and 3E shows GM-CSF level following intrauterine infusion of 4 μg GM-CSF mRNA in LNP C or 3.6 μg mouse recombinant GM-CSF in PBS at day 5 p.c in ( FIG.3D) the injected uterine horn after lavage and ( FIG.3E) plasma. Uteruses were flushed with PBS to remove uninternalized protein before tissue processing for ELISA. Data represent the mean ± SEM (n=3). FIG.3F shows uterus-to-plasma GM-CSF levels. FIGs.4A-4F show the therapeutic utility of GM-CSF mRNA in a uterine-factor infertility model. Figure 4A is a schematic of the induction of thin-endometrium (TE) in mice and treatment schedule. FIG.4B shows the GM-CSF production following intrauterine infusion of 4 μg GM-CSF mRNA-loaded LNP C in the TE uterus. LNP administration during the WOI was carried out on day 5 p.c. Outside the WOI, treatment was administered to unmated female mice. Data represent the mean ± SEM (n=6). Statistical analysis was performed using Fisher's LSD test. FIG.4C shows the numbers of embryo implantation sites in each injured and un-injured horn of treated and untreated groups. Data represent the mean ± SEM (n=6-9). Statistical analysis was carried out using multiple paired t-tests. FIG.4D 6 JHU C 17814 PCT 095238 / 00572 shows the compiled data showing the total number of embryo implantation sites, with the data from un-injured uterine horns from all three groups pooled into one group. Data represent the mean ± SEM (n=6 for LNP C and recombinant protein groups, n=9 for PBS group, and n=21 for un-injured uteruses). ANOVA followed by Dunnett's test. FIG.4E shows the percentage of uteruses positive to at least one implantation as calculated from FIG.4F shows the average endometrial thickness. Evaluable uterus segments between implantation sites with a clear uterine lumen were reported. Data represent the mean ± SEM (n=4-9). ANOVA followed by Dunnett's test. FIGs. 5A and 5B show estimation of RGD density on LNP C. In FIG.5A, assuming that the average phospholipid surface area is 0.65 nm2as previously reported1, 100 lipids will occupy roughly 65 nm2of total planar surface area. Additionally, assuming that all C18-RGD will remain distributed equally on all surfaces of the LNPs close to the feeding ratio (5 mol%), 5 out of the 100 lipids will comprise C18-RGD. FIG.5B shows the average closest center-to-center between adjacent RGD peptides is estimated as 5.2 nm, and the longest distance is 7.3 nm. FIGs. 6A and 6B show the luciferase expression after intravenous injections. LNPs A-L containing 2 μg fLuc mRNA were intravenously administered by tail vein injection in unmated healthy female CD-1 mice. After 4 hr, mice were sacrificed, and luciferase expression was quantified in (FIG. 6A) the spleen and (FIG. 6B) the whole uterus. Data represent the mean ± SEM (n=3 for all LNPs, except n=6 for LNP C). FIGs.7A and 7B: 2μg fLuc loaded-control LNP (LNP A) and RGD-LNP (LNP C) were administered intravenously by tail vein injections. (FIG.7A) The total luciferase expression 4 hr post-injection in tissue homogenates, and (FIG.7B) luciferase expression normalized to mg protein. Data represent the mean ± SEM (n=3). ***p<0.001, ****p<0.0001, n.s: non-significant. FIG.8 shows the ionization profile of LNPs. LNPs were incubated in buffers at pH spanning 4-9 followed by the quantification of TNS fluorescence using a microplate reader. The pKa was calculated as the pH value giving 50% maximal fluorescence intensity. Data represent the mean ± SEM (n=3). FIG.9 shows the impact of intrauterine infusions at day 5 p.c. on embryos.20 μL solution containing PBS or fLuc mRNA-loaded RGD-LNP (LNP C) was injected into the right horn. The number of resorbed (aborted) embryos was quantified visually on day 15 p.c. Data represent the mean ± SEM (n=4). 7 JHU C 17814 PCT 095238 / 00572 DETAILED DESCRIPTION OF THE INVENTION The lining of the uterus (endometrium) undergoes cyclical physiological changes throughout the menstrual cycle. Endometrial dysfunction is the cause of various women’s health disorders, including infertility. mRNA delivery by lipid nanoparticles (LNPs) can be used to introduce therapeutic proteins locally, when used in combination with a strategy for the selective targeting of mRNA to the endometrium. The off-target leeching of mRNA-LNPs from the site of injection to the liver or spleen is a drawback. A strategy has been developed that selectively targets the endometrium during the implantation window and avoids off-target distribution, as a new class of infertility treatments. Results demonstrate that LNPs can penetrate uterine mucosa to deliver cargo such as functional nucleic acids for example, mRNA to the endometrium. Targeting of the LNPs ensures that the LNPs penetrate and release the mRNA primarily to the tissue associated with the peak time period for implantation, thereby providing a means to address infertility. A. DEFINITIONS “Active agent” as used herein refers to a physiologically or pharmacologically active substance that acts locally and / or systemically in the body. An active agent is a substance that is administered to a patient for the treatment (e.g., therapeutic agent), prevention (e.g., prophylactic agent), or diagnosis (e.g., diagnostic agent) of a disease or disorder. “Hydrophobic” as used herein refers to a non-polar molecule or part of a molecule that cannot form energetically favorable interactions with water molecules and therefore does not dissolve in water. “Hydrophilic” as used herein describes a polar molecule or part of a molecule that forms enough energetically favorable interactions with water molecules to dissolve readily in water. “Amphiphilic” as used herein describes a molecule having both hydrophobic and hydrophilic regions, such as in a phospholipid or a detergent molecule. “Effective amount” and “suitable amount” as used herein with respect to a therapeutic agent is at least the minimum concentration required to effect a measurable improvement or prevention of any symptom or a particular condition or disorder, to effect a measurable enhancement of life expectancy, or to generally improve patient quality of life. The effective amount may vary depending on such factors as the disease or condition being treated, the active agent(s) (e.g., particular targeted constructs, etc.) being administered, the size of the subject, or the severity of the disease or condition. With regard to cancer, an effective amount can refer to an amount of the active agent that reduces or inhibits tumor growth or tumor 8 JHU C 17814 PCT 095238 / 00572 burden. The effective amount can be in the context of the delivery systems disclosed herein. For example, in some embodiments, “therapeutically effective amount” refers to an amount of the therapeutic agent that, when incorporated into and / or onto particles described herein, produces some desired effect at a reasonable benefit / risk ratio applicable to any medical treatment. One of ordinary skill in the art may empirically determine the effective amount of a particular compound without necessitating undue experimentation. “Pharmaceutically acceptable” as used herein refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio. “Individual,” “host,” “subject,” and “patient” as used herein are used interchangeably to refer to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. The subject can be a human or veterinary patient. “Treatment” as used herein refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. Use of the term “about” is intended to describe values either above or below the stated value in a range of approx. + / - 10%; in other forms the values may range in value either above or below the stated value in a range of approx. + / - 5%. 9 JHU C 17814 PCT 095238 / 00572 II. COMPOSITIONS Compositions for targeted endometrial delivery of an active agent include functionalized lipid nanoparticles (LNPs) as the carrier for the active agent. The functionalized lipid nanoparticles are incorporated into a formulation suitable for intrauterine delivery in combination with a pharmaceutically acceptable carrier. A. Lipid Nanoparticles (“LNPs”) The LNP (also referred to herein as vehicles) are modified to include a targeting moiety / ligand, typically conjugated to a lipid that inserts into, or otherwise forms part of, the LNP. Such targeting moiety / ligand can be added to the vesicle using, for example synthetic techniques that are known in the art. The LNP of the compositions provided herein, in some embodiments, have a mean geometric diameter that is less than 500 nm. In some embodiments, the nanocarriers have mean geometric diameter that is greater than 50 nm but less than 500 nm. In some embodiments, the mean geometric diameter of a population of nanocarriers is about 60 nm, 75 nm, 100 nm, 125 nm, 150 nm, 175 nm, 200 nm, 225 nm, 250 nm, 275 nm, 300 nm, 325 nm, 350 nm, 375 nm, 400 nm, 425 nm, 450 nm, or 475 nm. In some embodiments, the mean geometric diameter is between 100-400 nm, 100-300 nm, 100-250 nm, or 100-200 nm. In some embodiments, the mean geometric diameter is between 60-400 nm, 60-350 nm, 60-300 nm, 60-250 nm, or 60-200 nm. In some embodiments, the mean geometric diameter is between 75-250 nm. In some embodiments, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the nanocarriers of a population of nanocarriers have a diameter that is less than 500 nM, less than 400, less than 200 nm or between about 100 and 200 nm. In a preferred embodiment, the PDI range is >0 and <= 0.3 and E% >=70% and <100%. In some embodiments, the functionalized LNP has a negative zeta potential. In some embodiments, the LNP has a net negative charge at neutral pH. In some forms, the functionalized LNP has an increase in negative surface charge when compared to the non- functionalized LNP, by at least 30%, 40%, 50%, 60%, 70% etc, although the charge could also be reduced. Depending on the type of ligand chemistry, the change could be positive. The TL-functionalized LNPs typically include one or a combination of two or more lipids that can be neutral, anionic, or cationic at physiologic pH. Ionizable lipids are a class of lipid molecules which remain neutral at physiological pH, but are protonated at low pH, making them positively charged. Cationic lipids are amphiphilic molecules, which consist of a hydrophilic and a hydrophobic region connected by a linker structure. 10 JHU C 17814 PCT 095238 / 00572 Suitable neutral and anionic lipids include, but are not limited to, sterols and lipids such as cholesterol, phospholipids, lysolipids, lysophospholipids, sphingolipids or pegylated lipids. Neutral and anionic lipids include, but are not limited to, phosphatidylcholine (PC) (such as egg PC, soy PC), including, but limited to, 1 ,2-diacyl-glycero-3-phosphocholines; phosphatidylserine (PS), phosphatidylglycerol, phosphatidylinositol (PI); glycolipids; sphingophospholipids such as sphingomyelin and sphingoglycolipids (also known as 1- ceramidyl glucosides) such as ceramide galactopyranoside, gangliosides and cerebrosides; fatty acids, sterols, containing a carboxylic acid group for example, cholesterol; 1 ,2-diacyl- sn-glycero-3-phosphoethanolamine, including, but not limited to, 1 ,2- dioleylphosphoethanolamine (DOPE), 1 ,2-dihexadecylphosphoethanolamine (DHPE), 1 ,2- distearoylphosphatidylcholine (DSPC), 1 ,2-dipalmitoyl phosphatidylcholine (DPPC), and 1 ,2-dimyristoylphosphatidylcholine (DMPC). The lipids can also include various natural (e.g., tissue derived L-α-phosphatidyl: egg yolk, heart, brain, liver, soybean) and / or synthetic (e.g., saturated and unsaturated 1,2-diacyl-sn-glycero-3-phosphocholines, 1-acyl-2-acyl-sn-glycero- 3-phosphocholines, 1,2-diheptanoyl-SN-glycero-3-phosphocholine) derivatives of the lipids. Suitable cationic lipids include, but are not limited to, N-[1-(2,3-dioleoyloxy)propyl]- N,N,N-trimethyl ammonium salts, also referenced as TAP lipids, for example methylsulfate salt. Suitable TAP lipids include, but are not limited to, DOTAP (dioleoyl-), DMTAP (dimyristoyl-), DPTAP (dipalmitoyl-), and DSTAP (distearoyl-). Other cationic lipids also include, but are not limited to, dimethyldioctadecyl ammonium bromide (DDAB), 1 ,2- diacyloxy-3-trimethylammonium propanes, N-[1-(2,3-dioloyloxy)propyl]-Ν,Ν-dimethyl amine (DODAP), 1 ,2-diacyloxy-3-dimethylammonium propanes, N-[1-(2,3- dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1 ,2-dialkyloxy-3- dimethylammonium propanes, dioctadecylamidoglycylspermine (DOGS), 3 -[N-(N',N'- dimethylamino-ethane)carbamoyl]cholesterol (DC-Chol); 2,3-dioleoyloxy-N-(2- (sperminecarboxamido)-ethyl)-N,N-dimethyl-1-propanaminium trifluoro-acetate (DOSPA), β-alanyl cholesterol, cetyl trimethyl ammonium bromide (CTAB), diC14-amidine, N-ferf- butyl-N'-tetradecyl-3-tetradecylamino-propionamidine, N-(alpha- trimethylammonioacetyl)didodecyl-D-glutamate chloride (TMAG), ditetradecanoyl-N- (trimethylammonio-acetyl)diethanolamine chloride, 1 ,3-dioleoyloxy-2-(6-carboxy-spermyl)- propylamide (DOSPER), and N , N , N' , N'-tetramethyl- , N'-bis(2-hydroxylethyl)-2,3- dioleoyloxy-1 ,4-butanediammonium iodide. In some embodiments, the cationic lipids can be 1-[2-(acyloxy)ethyl]2-alkyl(alkenyl)-3-(2-hydroxyethyl)-imidazolinium chloride derivatives, for example, 1-[2-(9(Z)-octadecenoyloxy)ethyl]-2-(8(Z)-heptadecenyl-3-(2- 11 JHU C 17814 PCT 095238 / 00572 hydroxyethyl)imidazolinium chloride (DOTIM), and 1-[2-(hexadecanoyloxy)ethyl]-2- pentadecyl-3-(2-hydroxyethyl)imidazolinium chloride (DPTIM). In some embodiments, the cationic lipids can be 2,3-dialkyloxypropyl quaternary ammonium compound derivatives containing a hydroxyalkyl moiety on the quaternary amine, for example, 1 ,2-dioleoyl-3- dimethyl-hydroxyethyl ammonium bromide (DORI), 1 ,2-dioleyloxypropyl-3-dimethyl- hydroxyethyl ammonium bromide (DORIE), 1 ,2-dioleyloxypropyl-3-dimetyl-hydroxypropyl ammonium bromide (DORIE-HP), 1 ,2-dioleyl-oxy-propyl-3-dimethyl-hydroxybutyl ammonium bromide (DORIE-HB), 1 ,2-dioleyloxypropyl-3-dimethyl-hydroxypentyl ammonium bromide (DORIE-Hpe), 1 ,2-dimyristyloxypropyl-3-dimethyl-hydroxylethyl ammonium bromide (DMRIE), 1 ,2-dipalmityloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DPRIE), and 1 ,2-disteryloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DSRIE). In some forms, the TL is directly functionalized onto a lipid, such as a phospholipid, on the LNP. The TL for example, RGD, can be attached directly to a phospholipid (such as DSPE) on the LNP, or it can include a 2000 g / mol PEG spacer between the TL and the phospholipid. RGD peptide (Arg-Gly-Asp) can be conjugated directly to a phospholipid forming the backbone of the LNP (for example, DSPE) or indirectly using a PEG linker. For example, the ITL, such as RGD can be attached to the lipid at the aspartic acid moiety by a maleimide linker. The other components of LNPs are the “helper lipids,” which are phospholipids and cholesterol. In some forms, the phospholipid is a phosphatidylcholine (PC), such as 1,2- distearyol-sn-glycero-3-phosphocholine (DSPC) and hydrogenated soybean PC (HSPC). In some embodiments, the disclosed LNPs include or are formed of one or more of 1, 2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), 1, 2-distearoyl-sn¬-glycero-3- phosphatidylethanolamine (DSPE), and 1, 2-distearoyl-sn-glycero-3¬-phosphoethanolamine– N-[poly (ethyleneglycol) 2000 (DSPE–PEG) and can include a sterol. Such a sterol component may be selected from cholesterol or its derivative e.g., ergosterol or cholesterolhemisuccinate, but it is preferably cholesterol. The LNPs include a synthetic polymer such as poly-(ethylene glycol) (PEG) (see, e.g., Paphajopoulos, et al., PNAS, 88(24):11460-11464 (1991) doi: 10.1073 / pnas.88.24.11460). PEG is a water-soluble, low-immunogenicity, biocompatible polymer formed from ethylene glycol repeating units. They have many applications in medical and commercial settings, with molecular weights ranging from 200 to 35,000 g / mol. In some embodiments, the PEG is from about PEG 350 to about PEG 5000, or between about PEG 750 and about PEG 5000, or 12 JHU C 17814 PCT 095238 / 00572 between about PEG 1000 and PEG 2000. In a particular embodiment, the PEG is PEG 350. The range of PEG commonly used in LNPs is 350, 550, 750, 1000, 2000, 3000, 5000, preferably less than 2000. The lipid anchor in the PEG lipid can be a C14-C18 phospholipid. Exemplary PEG- Lipids include, but are not limited to: C14-PEG2000: 1,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy-(polyethylene glycol)-2000]; C14-PEG350: 1,2- dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy-(polyethylene glycol)-350]; C14-PEG1000: 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy- (polyethylene glycol)-1000]; C14-PEG3000: 1,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy-(polyethylene glycol)-3000]; C18-PEG2000: 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy-(polyethylene glycol)-2000]; C18-PEG350: 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy-(polyethylene glycol)-350], etc. In some forms, the TL-PL is at a concentration from about 1 to about 10 mol% of the total lipid concentration in the LNPs. In some forms, the TL-PL includes a linker between the integrin-targeting ligand (ITL) and the phospholipid (PL). In some form, no linker is present between the TL and the PL in the ITL-PL. The functionalized Lipid nanoparticles (“LNPs”) are preferably a 4-component ionizable LNPs composed of an ionizable lipid (such as D-Lin-MC3-DMA), helper lipid (such as cholesterol), phospholipid (such as DSPC), and a PEG-lipid (varying in PEG Mw and lipid tail length). Thus, in some forms, the LNPs include 4 components: ionizable lipid (IL), helper lipid (HL), an integrin targeting ligand functionalized phospholipid (ITL-PL), and a polyethylene glycol-Lipid (PEG-lipid). In some forms, the functionalized LNP includes an ionizable lipid (for example, 50 mol% D-Lin-MC3-DMA), helper lipid (for example, 38.5 mol% cholesterol), phospholipid (for example, 10 mol% DSPC), and a PEG-lipid (for example, 1.5 mol% DMG-PEG2000, DSPE-PEG350, or DSPE-PEG2000). 13 JHU C 17814 PCT 095238 / 00572 B. Targeting Ligands The LNP typically includes one or more a functional elements conjugated to or otherwise covalently linked, directly (without the presence of an intervening molecule serving as a linker) or indirectly (via an intervening molecule serving as a linker, to a lipid (also referred to as the lipid component of the LNP). “Covalent linkage” refers to a bond or organic moiety that covalently links functional elements to a surface of a LNP. The functional element can be a small molecule, protein or polypeptide, carbohydrate, nucleic acid or a combination thereof. In preferred embodiments, at least one of the functional elements is a targeting moiety that increases attachment, binding, or association of the functionalized LNP to target cell(s), tissues(s), and / or microenvironment(s) relative to the lipid vesicle, for example, endometrial cells, tissue, microenvironment, or integrin expressing cells. Additionally or alternatively the targeting moiety can increase attachment, binding, or association of the functionalized LNP to a target cell(s), tissues(s), and / or microenvironment(s) relative non-targeted cell(s), tissue(s), and / or microenvironment(s). This, in some forms, the one or more a functional elements is directly covalently linked to a lipid component on the LNP. When a linker is employed, in some forms the linker can be a PEG molecule. Any of a variety of methods can be used to associate a functional element or linker with the LNP. General strategies include passive adsorption (e.g., via electrostatic interactions), multivalent chelation, high affinity non-covalent binding between members of a specific binding pair, covalent bond formation, etc. In some embodiments, click chemistry can be used to associate a linker with a particle (e.g. Diels-Alder reaction, Huigsen 1,3- dipolar cycloaddition, nucleophilic substitution, carbonyl chemistry, epoxidation, dihydroxylation, etc.). A bifunctional cross-linking reagent can be employed. Such reagents contain two reactive groups, thereby providing a means of covalently associating two target groups. The reactive groups in a chemical cross-linking reagent typically belong to various classes of functional groups such as succinimidyl esters, maleimides, and pyridyldisulfides. Exemplary cross-linking agents include, e.g., carbodiimides, N-hydroxysuccinimidyl-4- azidosalicylic acid (NHS-ASA), dimethyl pimelimidate dihydrochloride (DMP), dimethylsuberimidate (DMS), 3,3'-dithiobispropionimidate (DTBP), N-Succinimidyl 3-[2- pyridyldithio]-propionamido (SPDP), succimidyl .alpha.-methylbutanoate, biotinamidohexanoyl-6-amino-hexanoic acid N-hydroxy-succinimide ester (SMCC), succinimidyl-[(N-maleimidopropionamido)-dodecaethyleneglycol]ester (NHS-PEO12), etc. 14 JHU C 17814 PCT 095238 / 00572 For example, carbodiimide-mediated amide formation and active ester maleimide-mediated amine and sulfhydryl coupling are widely used approaches. In some forms the one or more functional elements is an integrin-binding ligand (herein also, an integrin targeting ligand). The integrin binding peptides can be derived from collagen, fibronectin, and / or laminin. In some forms, the integrin binding peptide is RGD. Integrin binding peptides are disclosed for example in U.S. Patent No.5,912,234, for example, CRGDCL (SEQ ID NO:1), CRGDCA (SEQ ID NO:2); GACRGDCLGA (SEQ ID NO:3), NGRAHA (SEQ ID NO:4); CVLNGRME (SEQ ID NO:6), RCDVVV (SEQ ID NO:7), SLIDIP (SEQ ID NO:8), and TIRSVD (SEQ ID NO:9). Thus, the LNP are functionalized with an integrin targeting ligand, for example RGD peptide (Arg-Gly-Asp), conjugated directly to a lipid component of the LNP, such as DSPE or indirectly via a PEG. Targeting ligands can be added at between about 0.5 to about 10 mol% of the total lipid concentration in the LNP, in some forms from about 1.5 to about 7 mol% of the total lipid concentration, in some forms, from about 3 to about 6 mol% of the total lipid concentration in the LNP. In some embodiments, the targeting moiety targets cancer cells, particular cancer cells derived from endometrial cells. Examples include, but are not limited to HER2-targeted antibodies (HER2 is often overexpressed in endometrial cancer); folic acid, which can target endometrial cancer cells which often overexpress the folate receptor and anti-trophoblast surface antigen-2 specific antibodies for example, the humanized RS7 antibody targeting Trop-2 (USP 7238785, incorporated herein by reference). In other embodiments, the functional element is a detectable label such as a fluorophore, radiolabel, magnetic label, or a contrast agent. In some forms, the LNP includes a combination of functional elements such as an ITL, a cancer cell targeting moiety and / or a detectable label. C. Active Agents (Therapeutic and Prophylactic Agents) Agents to be delivered include therapeutic, nutritional, diagnostic, and prophylactic compounds. Proteins, peptides, carbohydrates, polysaccharides, nucleic acid molecules, and organic molecules, as well as diagnostic agents, can be delivered. One or more active agents may be formulated alone or with excipients or encapsulated on, in or incorporated into the nanocarriers. Active agents include therapeutic, prophylactic, nutraceutical and diagnostic agents. Any suitable agent may be used. These include organic compounds, inorganic compounds, proteins, polysaccharides, nucleic acids or other materials that can be incorporated using standard techniques. 15 JHU C 17814 PCT 095238 / 00572 Therapeutic agents include antibiotics, antivirals, anti-parasites (helminths, protozoans), anti-cancer (referred to herein as "chemotherapeutics", including cytotoxic drugs such as doxorubicin, cyclosporine, mitomycin C, cisplatin and carboplatin, BCNU, 5-FU, methotrexate, adriamycin, camptothecin, epothilones A-F, and taxol), antibodies and bioactive fragments thereof (including humanized, single chain, and chimeric antibodies), peptide drugs, anti-inflammatories, nutraceuticals such as vitamins, and nucleic acid drugs (including DNA, RNAs including mRNAs, antisense, siRNA, miRNA, anti-miRNA, piwi- interacting RNA (piRNA), aptamers, ribozymes, external guide sequences for ribonuclease P, and triplex forming agents such as “tail-clamp” peptide nucleic acids (tcPNAs)). In some embodiments, the active agent is a vector, plasmid, or other polynucleotide encoding a nucleic acid such as those discussed above. In some embodiments, the active agent is one or more nucleic acid molecules selected from mRNA (encoding a therapeutic and for delivering the encoded therapeutic) antisense, siRNA, miRNA, anti-miRNA, primary transcript miRNA (pri-miRNA), aptamers, ribozymes, external guide sequences for ribonuclease P, triplex forming agents, and CRIPSR / Cas component(s), or a polynucleotide encoding any of the foregoing. The miRNA can be a pri- miRNA, precursor miRNA (pre-miRNA), mature miRNA, miRNA mimic, or a fragment or variant thereof that retains the biological activity of the miRNA. In some embodiments, the nucleic acid such as miRNA, targets an oncogene. The active agent can be selected from cytokines (such as GM-CSF (142 nucleotides)), growth factors (hGH), genome editors (Cas9, Cre recombinase, etc), base / prime editors, therapeutic antibodies (anti-PD-1, immunotherapies, etc). mRNA can be nucleoside unmodified, and sometimes modified with N1-methylpseudouridine. In some instances, 5moU (5-methoxyuridine) is used to modify mRNA. The term growth factor corresponds to a family of secreted signaling proteins capable of inducing proliferation and differentiation in cells. Growth factors typically act as initiators of signaling cascades in cells by paracrine and endocrine interactions. Examples are cytokines and hormones that bind to specific receptors on the surface of their target cells, typically initiating a variety of cellular processes. They often promote cell differentiation and maturation, whose specificity varies among growth factors. For example, bone morphogenic proteins stimulate bone cell differentiation, and fibroblast growth factors and vascular endothelial growth factors stimulate blood vessel differentiation (vasculogenesis and angiogenesis). 16 JHU C 17814 PCT 095238 / 00572 Cytokines, a family of soluble proteins, were thought to be produced only in the bone marrow, immune system, and circulating blood cells when first identified. Later, it was shown that these proteins are produced in many tissues including reproductive and embryonic tissues. The term growth factor is sometimes used interchangeably with the term cytokine. Whereas growth factor implies a positive effect on cell division, cytokine is a neutral term with respect to whether a molecule affects proliferation. In this sense, some cytokines can be growth factors, such as granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor. However, some cytokines have an inhibitory effect on cell growth or proliferation. Yet others, such as the Fas ligand, are used as “death” signals; they cause target cells to undergo programmed cell death, or apoptosis. Growth factors can be classified according to both structural and evolutionary associations, ordering them into larger families of proteins. Recent years have seen a great increase in the number of growth factors identified and growth factor families such as bone morphogenic protein (BMP), fibroblast growth factor (FGF), transforming growth factor beta (TGF-β), neurotrophins (nerve growth factor [NGF], brain-derived neurotrophic factor [BDNF], and neurotrophin [NT3]), colony stimulating factors (CSF), platelet-derived growth factor (PDGF), erythropoietin (EPO), thrombopoietin (TPO), myostatin (GDF-8), growth differentiation factor 9 (GDF-9), epidermal growth factors (EGFs), hepatocyte growth factor (HGF), and more. Their role in implantation is becoming increasingly well understood. In some embodiments, the functional element include or is a tracking, imaging, or diagnostic moiety. Exemplary agents include paramagnetic molecules, fluorescent compounds, magnetic molecules, and radionuclides, x-ray imaging agents, and contrast agents. Exemplary materials include, but are not limited to, metal oxides, such as iron oxide, metallic particles, such as gold particles, etc. Biomarkers can also be conjugated to the surface for diagnostic applications. For imaging, radioactive materials such as Technetium99 (99mTc) or magnetic materials such as Fe2O3could be used. Examples of other materials include gases or gas emitting compounds, which are radioopaque. The most common imaging agents for brain tumors include iron oxide and gadolinium. Diagnostic agents can be radioactive, magnetic, or x-ray or ultrasound-detectable. Other detectable labels include, for example, radioisotopes, fluorophores (e.g., fluorescein isothiocyanate (FITC), phycoerythrin), enzymes (e.g., alkaline phosphatase, horseradish peroxidase), element particles (e.g., gold particles) or contrast agents. 17 JHU C 17814 PCT 095238 / 00572 For example, a fluorescent label can be chemically conjugated to a lipid to yield a fluorescently labeled lipid as exemplified below. In other embodiments the label is a contrast agent. A contrast agent refers to a substance that enhances the contrast of structures or fluids within the body in medical imaging. Contrast agents are known in the art and include, but are not limited to agents that work based on X-ray attenuation and magnetic resonance signal enhancement. Suitable contrast agents include iodine and barium. The methods of making the functionalized LNPs can include loading the LNPs or functionalized LNPs with an active agent. The loading of the functionalized LNPs typically includes mixing LNP components or LNPs and active agent alone or in combination with incubation, freeze-thaw cycling, sonication, extrusion, chemical transfection, electroporation, or a combination thereof. For example, where the active agent cargo is a nucleic acid molecule such as mRNA, LNPs can be prepared by rapid mixing (vortex or microfluidic mixer such as Nanoassemblr Ignite), by mixing mRNA in 50 mM sodium citrate buffer (pH=3) and ethanol phase containing the lipids. The nitrogen of amino lipids to the phosphate groups of mRNA molar ratio (N / P) can be fixed, for example at 6. Final LNP formulation is suspended in PBS. Particle size range is about 100-200 nm. D. Pharmaceutical Formulations Pharmaceutical compositions including the functionalized LNPs are also provided. The formulations are designed to provide maximum uptake in the affected tissues with rapid dissemination throughout the region to be treated, with little to no increase in systemic blood levels of the active agent. In some preferred forms, the pharmaceutical formulations are for transmucosal delivery, vaginally. In certain embodiments, the compositions are administered locally, for example, by injection directly into a site to be treated. Formulations for administration to the mucosa will typically be spray dried drug particles, which may be incorporated into a tablet, gel, capsule, suspension or emulsion. Standard pharmaceutical excipients are available from any formulator. The active agent may be "associated" in any physical form with the ITL-LNPs, for example, adsorbed or absorbed, adhered to or encapsulated in, and / or suspended or dissolved in a carrier such as an ointment, gel, paste, lotion, or spray. In some embodiments, the ITL- LNP packages two, three, four, or more different active agents for simultaneous delivery to a cell. 18 JHU C 17814 PCT 095238 / 00572 A "cream" is a viscous liquid or semi-solid emulsion of either the “oil-in-water” or “water-in-oil type”. An "emulsion" is a composition containing a mixture of non-miscible components homogenously blended together. “Gel” as used herein is a colloid in which the dispersed phase has combined with the continuous phase to produce a semisolid material, such as jelly. Tablets can be made using compression or molding techniques well known in the art. Gelatin or non-gelatin capsules can prepared as hard or soft capsule shells, which can encapsulate liquid, solid, and semi-solid fill materials, using techniques well known in the art. “Diluents”, also referred to as "fillers," are typically necessary to increase the bulk of a solid dosage form so that a practical size is provided for compression of tablets or formation of beads and granules. Suitable diluents include, but are not limited to, dicalcium phosphate dihydrate, calcium sulfate, lactose, sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose, kaolin, sodium chloride, dry starch, hydrolyzed starches, pregelatinized starch, silicone dioxide, titanium oxide, magnesium aluminum silicate and powdered sugar. “Binders” are used to impart cohesive qualities to a solid dosage formulation, and thus ensure that a tablet or bead or granule remains intact after the formation of the dosage forms. Suitable binder materials include, but are not limited to, starch, pregelatinized starch, gelatin, sugars (including sucrose, glucose, dextrose, lactose and sorbitol), polyethylene glycol, waxes, natural and synthetic gums such as acacia, tragacanth, sodium alginate, cellulose, including hydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose, and veegum, and synthetic polymers such as acrylic acid and methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, aminoalkyl methacrylate copolymers, polyacrylic acid / polymethacrylic acid and polyvinylpyrrolidone. “Lubricants” are used to facilitate tablet manufacture. Examples of suitable lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, glycerol behenate, polyethylene glycol, talc, and mineral oil. “Disintegrants” are used to facilitate dosage form disintegration or "breakup" after administration, and generally include, but are not limited to, starch, sodium starch glycolate, sodium carboxymethyl starch, sodium carboxymethylcellulose, hydroxypropyl cellulose, pregelatinized starch, clays, cellulose, alginine, gums or cross linked polymers, such as cross- linked PVP (Polyplasdone® XL from GAF Chemical Corp). “Stabilizers” are used to inhibit or retard drug decomposition reactions, which include, by way of example, oxidative reactions. Suitable stabilizers include, but are not limited to, 19 JHU C 17814 PCT 095238 / 00572 antioxidants, butylated hydroxytoluene (BHT); ascorbic acid, its salts and esters; Vitamin E, tocopherol and its salts; sulfites such as sodium metabisulphite; cysteine and its derivatives; citric acid; propyl gallate, and butylated hydroxyanisole (BHA). III. Methods of Treatment using Targeted LNPs The formulations are administered to a subject in need thereof, preferably, locally, to treat an upper urogenital tract infection / disease via spatiotemporal targeting. The formulations are preferably administered locally within the region to be treated, for example, vaginally for treatment of diseases of the ovaries and uterus. As used herein, "locally" can refer to topical application generally to the mucosal or endometrial surfaces of the vagina and / or uterus, or to a particular portion of the vagina or uterus. As used herein, "regionally" refers to reproductive organs and their surrounding environs, which include uterus, fallopian tube, peritoneal space, pelvic cul-de-sac, ovaries, perineum, and the rectovaginal region. As used herein, "systemically" refers to the circulatory system, and regions outside the spaces described above. The formulations are preferably not administered systemically, such as intravenously. In some embodiments, the compositions are delivered locally to the appropriate cells by using a catheter or syringe. Other means of delivering such compositions locally to cells include using infusion pumps (for example, from Alza Corporation, Palo Alto, Calif.) or incorporating the compositions into polymeric implants (see, for example, P. Johnson and J. G. Lloyd-Jones, eds., Drug Delivery Systems: Fundamentals and Techniques (Chichester, England: Ellis Horwood Ltd., 1988 ISBN-10: 0895735806), which can effect a sustained release of the drug to the immediate area of the implant. Vaginally administered pharmaceutical preparations as described herein are particularly effective in treating certain diseases of female reproductive systems, such as endometriosis. Female UGT diseases affect a large number of women each year. For example, pelvic inflammatory disease (PID), which includes endometritis, parametritis, chorioamnionitis (infection of the placental membrane), salpingitis, oophoritis, peritonitis, tubo-ovarian and pelvic abscess and septicemia, is a common cause of gynecologic hospitalization. As reported by Patel, et al. Adv. Drug Del. Rev 177, 113955 (2021), the prevalence of PID in the United States alone is an estimated 2.5 million women, aged 18–44 years. Pathogens residing in the vagina and cervix, including Chlamydia trachomatis, Neisseria gonorrhoeae, and Mycoplasma genitalium, and those involved in bacterial vaginosis and inflammatory vaginitis, can ascend to the UGT causing inflammation of reproductive and pelvic organs, 20 JHU C 17814 PCT 095238 / 00572 including the fallopian tubes, uterus, ovaries, and peritoneum. These infections can manifest as abdominal pain and may lead to tubal infertility, ectopic pregnancy, pre-term birth (PTB) or transmission of infections to infants during childbirth. Moreover, pathologies within the UGT can traverse into the systemic circulation given the access to abdominal organs via the UGT. Similarly, many pathological conditions of UGT can substantially impact a woman’s fertility. A commonly diagnosed UGT disease is uterine leiomyoma (uterine fibroids), a non- cancerous growth within the myometrium. The cumulative incidence of uterine fibroids nears 70% in white women and >80% in black women by age 50 years, with an estimated annual burden of $5.9–34.4 billion to the United States. Similarly, intrauterine adhesions (IUAs), adenomyosis, polycystic ovarian syndrome (PCOS), preeclampsia (PE) and postmenopausal syndrome are other commonly diagnosed issues with substantial impact on fertility, PTB, morbidity, pelvic pain, and mental health disorders. Gynecologic cancers are one of the major disease categories that contribute to high mortality among adult women of all age groups. Endometriosis The endometrium is the mucosal lining of the uterine cavity that consists of a mucus layer, epithelial cells, and a stroma (fibroblasts). It is sensitive to hormones like estrogen and progesterone, and during each menstrual cycle, the body grows a new endometrium to prepare for a fertilized egg. The endometrium thickens and becomes rich in blood vessels and glands, creating an ideal environment for a blastocyst to implant in the uterus. If pregnancy doesn't occur, the endometrium is shed during menstruation and then regenerates during subsequent non-pregnancy cycles and after giving birth. Endometriosis is a chronic inflammatory disease characterized by the presence of ectopic endometrial tissues. It can cause severe pain in the pelvis and make it harder to get pregnant. The estimated global prevalence of endometriosis is approximately 10% of all reproductive-age women. Endometriosis can start at a person’s first menstrual period and last until menopause. With endometriosis, tissue similar to the lining of the uterus grows outside the uterus. This leads to inflammation and scar tissue forming in the pelvic region and (rarely) elsewhere in the body. The cause of endometriosis is unknown. There is no known way to prevent endometriosis. There is no cure, but its symptoms can be treated with medicines or, in some cases, surgery. It causes a chronic inflammatory reaction that may result in the formation of scar tissue (adhesions, fibrosis) within the pelvis and other parts of the body. Several lesion 21 JHU C 17814 PCT 095238 / 00572 types have been described: superficial endometriosis found mainly on the pelvic peritoneum, cystic ovarian endometriosis (endometrioma) found in the ovaries, deep endometriosis found in the recto-vaginal septum, bladder, and bowel and in rare cases, endometriosis has also been found outside the pelvis. Endometrial Cancer Uterine cancer includes two types of cancer: endometrial cancer (more common) and uterine sarcoma (rare). Uterine cancer symptoms include bleeding between periods or after menopause. Treatment often consists of a hysterectomy to remove your uterus. Endometrial cancer is the most prevalent gynecologic malignancy in American women and a significant cause of morbidity and mortality. Chemotherapy is generally recommended for advanced or high-risk endometrial cancers, including those with spread beyond the uterus or high-grade histology. A regimen of carboplatin and paclitaxel is the most commonly used chemotherapy for endometrial cancer, either concurrently or sequentially with radiation therapy. In some cases, chemotherapy alone may be used. Checkpoint inhibitors (e.g., pembrolizumab and atezolizumab) effectively treat tumors with mismatch repair deficiency. For patients with serous endometrial cancers with over-expression of human epidermal growth factor receptor 2 (HER2), which contributes to cancer cell growth and proliferation, anti-HER2 medications target the mutation and are often added to standard chemotherapy to improve survival outcomes. immunotherapy to block the programmed death-1 pathway, which, if left unchecked, represses cytotoxic immune responses, was associated with improved responses in patients with MMRd endometrial cancers that are characterized by marked tumor- infiltrating lymphocytes, which are hallmarks of Lynch syndrome-linked cancers. The FDA approved pembrolizumab for MMRd endometrial cancer treatment, and in 2021, dostarlimab was also approved. Polyovarian Cystitis Polycystic ovary syndrome (PCOS) is a hormonal condition that can cause cysts to develop on the ovaries. These cysts are fluid-filled sacs, or follicles, that contain eggs that haven't been released from the ovary due to hormonal imbalances. Over time, the ovaries can fill with many small cysts. PCOS is a very common hormone problem for women of childbearing age. Women with PCOS may not ovulate, have high levels of androgens, and have many small cysts on the ovaries. PCOS can cause missed or irregular menstrual periods, excess hair growth, acne, infertility, and weight gain. Infertility and Fertility Treatments 22 JHU C 17814 PCT 095238 / 00572 As of 2021, globally, 9% of women of reproductive age including 1.5 million women in the United States, are infertile. The burden of infertility in developing countries can reach up to 30% of reproductive-aged women. Infertile women are 6–8 times more likely to have endometriosis The Examples show delivery of mRNA encoding for GM-CSF as a therapeutic intervention to regenerate endometrial thickness. Clinically, women with a thin endometrium (<7 mm) have a low rate of fertility. GM- CSF is mainly secreted from endometrial epithelium and increases endometrial thickness by regulating homeostasis and immune responses. GM-CSF is commonly administered in the clinic before embryo implantation during IVF to enhance endometrial receptivity. It can be administered as a single intra-uterine infusion or multiple subcutaneous injections. mRNA, however, can be beneficial to provide a transient yet a prolonged protein expression in the target tissue. mRNA is highly scalable and easy to synthesize and diversify by changing its sequence. In addition, multiple proteins encoding mRNAs can be delivered simultaneously in the same delivery vehicle. Therefore, introducing cytokine-encoding mRNA therapeutics can be used for infertility treatments. Implantation is the first step of crosstalk between the embryo and endometrium, which is the key point for a successful pregnancy. The implantation process includes apposition, adhesion, and invasion. Implantation can be broadly divided into three steps: apposition, attachment, and invasion. During apposition, numerous small pinopodes on the receptive endometrium developed by generalized stromal oedema on the inner uterine surface interlace with the microvilli on the outer surface of the cytotrophoblast. Heparin-binding EGF-like growth factor (HB-EGF) is expressed by the receptive endometrium, and cells that express transmembrane HB-EGF adhere to blastocysts displaying ErbB4 on their cell surface. Activated blastocysts upregulate their expression of HB-EGF, which via an auto-induction loop prompts its own gene expression in the endometrium at the site of blastocyst apposition. HB-EGF expression is modulated by Lif and results in a reduction of COX-2, the deficiency of which results in implantation failure. Gene expression in the Wnt / β-catenin pathway is also important at the site of implantation. Wnt / β-catenin signaling immediately before attachment requires activated blastocyst and preimplantation estrogen secretion in transgenic mice models. 23 JHU C 17814 PCT 095238 / 00572 In the attachment phase, various glycoproteins, carbohydrate ligands, receptors, and integrins work together to adhere the embryo to the endometrial surface. Several integrins are involved with implantation in varying capacities. During attachment α5β1, αvβ3, αvβ5, and αvβ6 are expressed by the embryo, and α1β1, α6β1, and α7β1 are subsequently involved in invasion. The ligand osteopontin of epithelial origin acts to bind integrin αvβ3 on the maternal surface to support adhesion. Further, during attachment, L-selectin molecules are presented on the blastocyst surface, and selectin oligosaccharide ligands are expressed in the primed endometrium. Interestingly, the integrins are dynamic with α5β1 starting within the inner cell mass in early embryo development and then translocated to the trophoblast cells that invade the endometrium during implantation. Invasion is dependent on endometrial vascular permeability, and decidualization is mediated by prostaglandin synthesis by COX1 and COX2. Various cellular molecules are essential for normal implantation and aberrant production or loss of them may be linked to unexplained infertility. In fact, implantation has been considered so dependent on these mediators that integrins have been suggested as potential biomarkers of infertility. As reviewed by Guzeloglu-Kayisli et al Semin Reprod Med.200927(1):62-79. Doi: 10.1055 / s-0028-1108011. Epub 2009 Feb 5. PMID: 19197806; PMCID: PMC3107839, Implantation, a critical step for establishing pregnancy, requires molecular and cellular events resulting in uterine growth and differentiation, blastocyst adhesion, invasion, and placental formation. Successful implantation requires a receptive endometrium, a normal and functional embryo at the blastocyst stage, and a synchronized dialogue between maternal and embryonic tissues. In addition to the well-characterized role of sex steroids, the complexity of embryo implantation and placentation is exemplified by the number of cytokines and growth factors with demonstrated roles in these processes. Disturbances in the normal expression and action of these cytokines result in an absolute or partial failure of implantation and abnormal placental formation in mice and human. Members of the gp130 cytokine family, interleukin- 11 (IL-11) and leukemia inhibitory factor, the transforming growth factor beta superfamily, the colony-stimulating factors, and the IL-1 and IL-15 systems are crucial molecules for a successful implantation. Chemokines are also important, both in recruiting specific cohorts of leukocytes to the implantation site and in trophoblast trafficking and differentiation. This review provides discussion of the embryonic and uterine factors that are involved in the process of implantation in autocrine, paracrine, and / or juxtacrine manners at the hormonal, cellular, and molecular levels. 24 JHU C 17814 PCT 095238 / 00572 Implantation necessitates complex interactions among the developing embryo, decidualizing endometrium, and developing maternal immune tolerance and / or alterations in cellular and humoral immune responses. All of these cellular and molecular events are controlled by endocrine hormones, including sex steroids and human chorionic gonadotropin (Hcg), and by local paracrine factors. Such factors are produced by the endometrial epithelium, decidualized stromal cells, and immune cells; they are secreted locally into the uterine lumen and the area of the decidual reaction, where they affect not only blastocyst development, attachment, and invasion, but also each other’s cellular functions and the transformation of the decidualizing extracellular matrix. These locally produced signaling molecules consist of growth factors, cytokines, and chemokines. Development of the embryo to the blastocyst stage, its implantation into the endometrium, and the formation of a functional placenta are essential steps in the establishment of pregnancy. Like many developmental processes, pregnancy involves a complicated series of genetic, molecular, and cellular interactions, all of which must be executed within an optimal time frame. In mammals, the fertilized egg undergoes many cell divisions to form a blastocyst, which is able to attach to the uterine epithelium. The implantation process starts when a free-floating blastocyst communicates with the endometrium. The adhesive interactions between the trophoblasts and the endometrial surface epithelium are followed by local invasion. Therefore, implantation requires the synchronous development of a blastocyst competent to implant and an endometrium able to respond to signals from the blastocyst. Countless endocrine, paracrine, and autocrine interactions during implantation occur among maternal-maternal, embryo-embryonic, and maternal-embryonic cells that mediate a complex dialogue between endometrium and the conceptus. These developmental events are orchestrated by sex steroids, Hcg, growth factors, cytokines, adhesion molecules, the extracellular matrix (ECM) proteins, and prostaglandins. Many crucial growth factors and cytokines affect blastocyst implantation and / or endometrial receptivity; however, the molecular mechanisms regulating this process in human is still poorly understood and needs further study to have clinical treatment options for infertility patients related to implantation failure or embryo rejection. The incidence of early pregnancy loss during or immediately after implantation is high, estimated at 25 to 40%. Of the pregnancies that are lost, most are lost very early and represent a failure of implantation. Failed implantation is also a major limiting factor in assisted reproduction. Implantation failure has been estimated to be related to inadequate endometrial receptivity in up to two thirds of cases. Endometriosis, endocrine abnormalities, thrombophilias, immunological 25 JHU C 17814 PCT 095238 / 00572 factors, and congenital and acquired anatomical factors may contribute to implantation failure. Implantation failure, or when a fertilized egg doesn’t implant in the uterine wall, can have many causes, including structural issues like uterine fibroids, polyps, septa, or adhesions, as well as menstrual cycle abnormalities and uterine congenital malformations. Uterine fibroids can block the fallopian tubes or prevent a fertilized egg from attaching to the uterus. Hormonal causes include hormonal imbalances, progesterone resistance, or changes in basal body temperature. Timing and Effective Amounts for Treatment The compositions are administered locally, at an effective time to ensure selective targeting of elevated integrin expression at the site of treatment. Spatiotemporal targeting entails that the strategy provided herein delivers effective amounts of one or more active agents, for example, mRNA to the endometrium specifically to the endometrium excluding the myometrium, liver, or spleen, lung, etc (spatial), and specifically during the implantation window in the reproductive cycle (temporal). The implantation window usually occurs on day 20-24 of the menstrual cycle in humans, or 6-10 days after ovulation. Thus, in some forms, the method includes administering an effective amount of active agent to the subject at day 20-24 of the menstrual cycle. In some forms the methods include determining the implantation window for the subject prior to administration of the compositions containing active agent. Methods for determining the window of implantation are known in the art (Enciso, et al., Sci Rep, 11:13420 (2021). The present invention will be further understood by reference to the following non- limiting example. EXAMPLES Example 1: SPATIOTEMPORAL MRNA TARGETING TO THE ENDOMETRIUM FOR THE TREATMENT OF INFERTILITY Materials and Methods Materials CleanCap® AG Firefly (fLuc), EGFP, and m1Ψ-modified mouse GM-CSF (CSF2) mRNA were purchased from Trilink Biotechnologies (San Diego, CA, USA). DSPC, DSPE- PEG2000 (C18-PEG2000), DMG-PEG2000 (C14-PEG2000), DSPE-PEG350 (C18- PEG350), and DMPE-PEG350 (C14-PEG350), DSPE-RGD (C18-RGD) were purchased from Avanti Research. D-Lin-MC3-DMA and Vitronectin Protein, Mouse (HEK293, His) 26 JHU C 17814 PCT 095238 / 00572 were purchased from MedChemExpress. Cholesterol was obtained from Sigma. DiD' solid was purchased from Invitrogen. DSPE-PEG2000-RGD (C18-PEG2000-RGD) was obtained from BOC Sciences. Mouse GM-CSF was obtained from Miltenyi Biotec. Luciferin substrate and reporter lysis buffer were purchased from Promega. FluoSpheres™ carboxylate-modified 0.1 μm polystyrene beads with a 580 / 605 fluorophore, the Pierce™ BCA Protein Assay Kit, and the Quant-itTM RiboGreen RNA Assay Kit were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Cy5 Label IT Nucleic Acid Labeling Kit was purchased from Mirus. A LipidLaunch™ LNP Apparent pKa Assay Kit was provided by Cayman. OCT compound was purchased from SciGen Ltd. (Singapore). Rabbit GFP Polyclonal Primary Antibody and Goat anti-Rabbit IgG conjugated to Alexa Fluor™ 488 were obtained from Thermo. Alexa Fluor® 647 Anti-pan Cytokeratin antibody (C-11), rabbit Alexa Fluor® 647 Anti-Vimentin antibody (EPR3776), and goat FITC Anti-GFP antibody (Ab6662) were purchased from Abcam. DAPI Fluoromount-G® was obtained from SouthernBiotech. LNP formulation and characterization LNPs were formulated by a rapid mixing method.8 volumes of an ethanolic solution containing D-Lin-MC3-DMA, DSPC, Cholesterol, PEG-lipid, and RGD-conjugated lipid was prepared and mixed with 1 volume of mRNA solution in 1 mM sodium citrate pH 6.4. The mixture was then mixed drop-wise into 2.66 volumes of 50 mM sodium citrate buffer pH 3 and let to equilibrate for 10 minutes at room temperature. The resulting mixture was then diluted in PBS and concentrated using 30kD Amicon centrifugal filters (Millipore, MA, USA) for buffer exchange and removing residual ethanol. The final mRNA concentration in LNPs was 100 or 200 µg / mL. For fluorescence tagging in multiple particle tracking, DiD was added to the lipid phase at a final concentration of 0.1 mol%. For quantifying LNP biodistribution, mRNA was labeled with Cy5 using the Mirus labeling kit. Encapsulation ratios were determined using the RiboGreen Assay. Particle size and the polydispersity index (PDI) were measured using dynamic light scattering (DLS) equipped with a diode laser (λ = 532 nm) with a scattering angle of 173◦ (Zetasizer Nano-ZS, Malvern Instruments, Worcestershire, UK) at 10x dilution in PBS. The ζ-potential was determined by laser-Doppler electrophoresis using the same Zetasizer Nano-ZS at 50x dilution in PBS. The degree of LNP protonation at different pH values was determined using the 6-(p-toluidino)-2-naphthalene sulfonic acid sodium salt (TNS) method. The pKa was calculated by non-linear regression using a four- parameter model. Animal welfare statement 27 JHU C 17814 PCT 095238 / 00572 All experimental procedures were approved by the Johns Hopkins University Animal Care and Use Committee. Johns Hopkins Program of Animal Care and Use is accredited by AAALAC International. Animal care and procedures follow the Guide for the Care and Use of Laboratory Animals 8th ed80. CD-1 mice were purchased from Charles River Laboratories at 6–8 weeks and were housed in a 12 light / 12 dark cycle room. Time-mated female mice were purchased from Charles River Laboratories and delivered on post coitus (p.c.) day 4 and allowed to acclimate until dosing on p.c. day 5. For the infertility model experiments, female and male CD-1 (8-12 weeks) mice were mated at a 1:1 ratio in individual cages. Male CD-1 mice were individually housed and acclimated for at least a week before mating with female mice. Successful insemination was confirmed by the observation of a vaginal plug. The day a vaginal plug was observed was assigned as p.c day 1. Multiple-particle tracking on endometrial surfaces To characterize the mobility of mRNA-LNPs after infusion injection in the uterine cavity, particle diffusion was visualized on the surface of excised mouse endometrial tissue. Around 2 mm of a mouse uterus (p.c. day 5) was excised, flattened, and placed on a slide with a cavity of sufficient depth to allow a coverslip to contact the surface without compressing the tissue, exposing the endometrial surface. DiD-labeled mRNA-LNP (0.5 µl, 5 ng mRNA) was carefully added to the surface of the endometrium to prevent any pooling or dilution. The sample was then covered with a glass cover slip and sealed with a mountant. Multiple-particle tracking analysis was carried out using a ZEISS Axiovert epifluorescence microscope equipped with a 100× oil immersion objective. Trajectories were analyzed in each measurement and the coordinates of LNP centroids were transformed into time-averaged mean square displacements (<MSD>) using Matlab software. The experiment was performed in triplicate, and 8-12 videos were acquired in each replicate, taking care to avoid any areas with tissue spreading or fluid convection. An average of 391 ± 144, 398 ± 149, and 335 ± 176 particles of polystyrene nanoparticle, control-LNP, and RGD-LNP were analyzed in each measurement, respectively. The % particle distribution histograms were fitted into a Gaussian model using a non-linear regression of 189 degrees of freedom to calculate the mean MSD at t = 1 sec. Intrauterine injection of reporter mRNA-LNPs Mice were induced in a chamber and maintained under isoflurane anesthesia using a nose cone system. The abdomen was swabbed with 10% Povidone-iodine solution and a midline laparotomy was performed. Using surgical microscopes, the right uterine horn was isolated, and 20 µL containing the mRNA-LNP (2 µg fLuc mRNA or EGFP mRNA) solution 28 JHU C 17814 PCT 095238 / 00572 was injected into the uterine horn using a 30 G needle. The successful injection was confirmed by the observation of a bulge in the uterus as a result of a fluid-filled lumen. A routine closure was performed using 5.0 polyglycolic acid sutures in a simple continuous pattern for the peritoneum, followed by staple application for skin closure. Short-acting buprenorphine analgesia (0.1 mg / kg) was injected subcutaneously near the incision site while the mouse was still under anesthesia. Mice were sacrificed 4 hr post-injection for the evaluation of luciferase expression in organs, or 24 hr for microscopic observation of EGFP tissue distribution. Luciferase expression and biodistribution analysis After the mice were sacrificed 4 hr post-treatment, the whole uterus, cervix, liver, and spleen were collected and placed in screw-cap tubes each containing 0.5 mL 1x reporter lysis buffer. The tissues were then homogenized after the addition of stainless-steel beads and shaken at 6 m / sec for 40 sec using MP Biomedical FastPrep-245G Homogenizer. The samples were subjected to a single freeze-thaw cycle at -80 C. The tubes were spun at 8000x g for 5 min and 4°C. A 20 µL of the supernatant was transferred into a 96-well flat clear bottom white microplate. Luminescence was measured after auto-injection of a 100 µL luciferin substrate into each well using a Tecan microplate reader. Data were presented as photons per second exposure. For the normalization of luciferase expression to mg protein in each sample, protein content was quantified using the BCA assay. Cy5 labeled mRNA LNPs were administered by tail vein injection for the evaluation of LNP biodistribution. After 1 hr post-injection, mice were sacrificed and organs were collected and imaged at excitation and emission wavelength 620 and 670, respectively using a Perkin-Elmer Lumina In Vivo Animal Imaging System (IVIS). Cryo-sectioning and Immunostaining Tissue fixation was carried out as described before26. Briefly, the extracted organs were immersed in 4% paraformaldehyde (PFA) in PBS at 4 ◦C overnight. The next day, organs were incubated in 10% sucrose in PBS for 4 h, 15% sucrose in PBS for 4 h, and 20% sucrose in PBS overnight at 4 ◦C. The organs were then embedded in OCT compound and frozen gradually at -80 ◦C. Serial cross-sections of the uterus were then prepared at 14 μm thickness using a Cryostat (Leica Biosystems, Wetzlar, Germany) and placed onto positively charged slides. For immunostaining, the sections were thoroughly washed and treated with a blocking buffer (2% BSA in PBS containing 0.1% w / v Tween 20) for 1 h at room temperature. EGFP was stained with a rabbit GFP primary antibody (Thermo) at 300x dilution in blocking buffer 29 JHU C 17814 PCT 095238 / 00572 at 4 ◦C overnight. The next day, the primary antibody was washed off and the sections were incubated with goat anti-Rabbit IgG conjugated to Alexa Fluor™ 488 at 500 x dilution for 1 hr. After the last washing step, the slides were stained with DAPI and observed using an Axiovert epifluorescence microscope or LSM 710 confocal laser scanning microscope (Zeiss, CA, USA). For co-staining with an epithelial cell marker, the sections were labeled with a mouse Alexa Fluor® 647 Anti-pan Cytokeratin antibody mixed at 300x dilution with the GFP primary antibody. For co-staining with a stromal cell marker, rabbit Alexa Fluor® 647 Anti- Vimentin antibody and goat FITC Anti-GFP antibody were both diluted at 300x and incubated at 4 ◦C overnight before microscopic evaluation as described above. Measurement of GM-CSF tissue levels by ELISA Mice were injected 20 µl containing 4 ug GM-CSF mRNA-loaded LNPs or 3.6 µg recombinant mouse GM-CSF in the right horn in un-mated or mated (p.c. day 5) CD-1 mice. At definite time points, mice were sacrificed and the organs were collected, weighted, and homogenized in 0.5 mL reporter lysis buffer, as described in section 4.6. The blood was collected in heparinized tubes from the inferior vena cava of mice under isoflurane anesthesia. The plasma was collected after centrifugation at 2000x g for 5 min at 4 ◦C and stored at -80 until analyzed. The blood plasma and the tissue homogenate supernatants were adequately diluted and assayed for the content of GM-CSF using a mouse GM-CSF ELISA kit according to the manufacturer’s protocol. To remove excess amounts of GM-CSF, the uterus lumen was flushed with 1 mL PBS before tissue processing for ELISA. Ethanol-injury model of thin endometrium A thin-endometrium (TE) model of uterine-factor infertility was induced by infusing the uteruses with ethanol as described in the literature52. Briefly, the uterine horns were exposed and isolated as described in section 4.5.25 µL 95% ethanol was injected into the right horn, while the left horn was ligated using a surgical clamp to prevent ethanol leakage into the control uterine horn. Extended-release buprenorphine (3.25 mg / kg) was injected at the site of surgery. Mice were then housed for 7 days for the TE to model. Treatment administration and evaluation of fertility rates Before treatment was administered, TE mice were mated with male mice and checked daily for the presence of a vaginal plug. On p.c. day 2, the abdomen was re-opened and 20 µL PBS, 4 µg GM-CSF mRNA-loaded LNP, or 3.6 µg recombinant mouse GM-CSF were administered to the ethanol-injured uterine horn. The left uterine horn was untreated and used as an internal control for successful implantation in each mouse. On p.c. day 7-9, mice were 30 JHU C 17814 PCT 095238 / 00572 sacrificed and the uteruses were extracted, imaged with digital photography, and the embryo attachment sites were counted. Histopathology Uterus segments that lie between embryo attachments were excised and fixed in 4% PFA at 4 ◦C overnight. Tissues were then sent to the JHMI Reference Histology core for paraffin embedding, cross-sectioning at 6 μm thickness, and staining with hematoxylin and eosin (H&E). Sections were imaged and analyzed using a Nikon light microscope. The endometrium thickness was calculated as the average of 4 measurements capturing the endometrial layer length extending from the uterine lumen to the myometrial layer. Evaluable uterine sections showing a clear uterine lumen without a decidual tissue at p.c. Days 7-9 were reported. Statistical analyses The statistical significance between the two groups was analyzed using an unpaired, two-tailed Student’s t-test. In the fertility experiment comparing embryo counts between the left and right uterine horns in the same mouse, a multiple-paired t-test was used. Multiple comparisons among three or more groups were performed using one-way or two-way ANOVA followed by Tukey’s post hoc test unless indicated otherwise. Comparison against untreated controls was performed using Dunnett’s test. The statistical significance in the % of treated animals showing at least 1 implantation sight was calculated using a 2X2 contingency table against the uninjured controls. A statistically significant difference was set at p<0.05. n.s: non-significant. 31 JHU C 17814 PCT 095238 / 00572 Results Preparation and characterization of mRNA-LNPs conjugated with RGD peptides Achieving multivalent interactions between targeting ligands and their receptors often leads to higher cellular uptake and delivery efficiency. Here, a broad affinity, linear RGD sequence was used to maximize the interaction with a wide range of integrins overexpressed on the endometrium during the WOI44. It was proposed that directly conjugating RGD to the DSPE lipid component (C18-RGD), rather than conventionally using a polyethylene (PEG) spacer, would allow for higher ligand densities on the LNP surface and enhance multivalent interactions with integrin receptors on the endometrium. However, a PEG-lipid stabilizer is needed in LNP formulations to ensure colloidal stability. To minimize steric hindrance and enhance RGD exposure on the LNP surface, the PEG component of the PEG-lipid stabilizer was shortened from the most commonly used 2000 g / mol to 350 g / mol. Specifically, a 350 g / mol PEG attached to DSPE (C18-PEG350) was used. fLuc mRNA-loaded RGD-LNPs co- formulated with 5 mol% of C18-RGD and 1.5 mol% C18-PEG350 were prepared. As a control, the same LNPs were formulated without adding the C18-RGD. Both LNPs showed comparable particle size, encapsulation ratios, and polydispersity indices (PDI) (Figure 1A). The ζ-potential of the RGD-LNP was more negative than the control-LNP, possibly due to the negative charge of the phosphatidylethanolamine moiety of C18-RGD (Figure 1A). Mucus is the first barrier for nanoparticle diffusion to the underlying endometrial tissues. Using multiple-particle tracking analysis, the mobility of control- and RGD-LNP on endometrial surfaces was characterized (Figures 1B-1D). In mice, embryo implantation occurs at post-coitus (p.c.) days 4-545. The integrin expression on mouse endometrium was previously reported to peak at day 5 p.c46,47. Here, uteruses were extracted from mice at day 5 p.c. and studied the diffusion of DiD-labeled mRNA-LNPs on the endometrial surface using multiple particle tracking analysis. Also observed was the diffusion of a fluorescently labeled carboxylate functionalized polystyrene nanoparticle (PS-COOH) as a control for poor mucus penetration due to nonspecific interaction with biological components48. Initially, it was determined the time-resolved changes in mean squared displacement (MSD) for both mRNA- LNPs and PS-COOH (Figure 1B). While the PS-COOH showed poor diffusion on the mouse endometrium as indicated by low MSD values, both control- and RGD-LNPs showed 40- and 80-fold increase in MSD at t= 1 sec compared to PS-COOH, respectively (p<0.0001, p<0.0006; Figure 1B). The RGD-LNP showed a 2-fold reduced MSD diffusion at t= 1 sec compared to the control-LNP (p=0.001, Figure 1B). Distribution histograms were created 32 JHU C 17814 PCT 095238 / 00572 based on MSD at time = 1 sec (Figure 1C). A Gaussian model was applied by nonlinear regression to estimate the mean Log10(MSD t = 1 sec). Both control- and RGD-LNPs showed around 2-fold higher mean Log10(MSD t = 1 sec) than PS-COOH (Figure 1C). Approximately 64% and 48% of particles in the control- and RGD-LNP groups showed mean Log10(MSD t = 1 sec) >-1 (MSD at t = 1 sec greater than 0.1), respectively, while only 11.6% of PS-COOH particles showed mean Log10(MSD t = 1 sec) >-1 (Figure 1D). The high mobility of mRNA- LNPs on the endometrial mucosa could be attributed to PEG and zwitterionic phospholipids on the LNP surface, which both could act as anti-fouling agents and enhance tissue penetration49. The slightly reduced diffusion of RGD-LNP compared to control LNP could be due to the interaction between RGD and integrin receptors on the surface of the endometrium, restricting particle diffusion slightly. Additionally, the anionic charge on RGD-LNP (Figure 1A) could reduce the mobility of the LNPs by inducing higher non-specific electrostatic interaction. RGD-LNPs induce high mRNA delivery to the endometrium when infused during the implantation window Next, control- and RGD-LNPs loaded with fLuc mRNA were infused into the uterine lumen of mice at day 5 p.c. (within the WOI). There was 3.9-fold higher luciferase expression in uteruses treated with RGD-LNP compared to the control LNP (Figure 1E). Additionally, there was >450- and 65-fold reduction in liver and spleen expression in the RGD-LNP treated group compared to the control-LNP, respectively (Figure 1E). To confirm the temporal targeting specificity of the RGD-LNP to the uterus during the WOI, control- and RGD-LNPs were infused in unmated female mice (i.e., outside the WOI). There was no significant difference in luciferase expression in uteruses between control- or RGD-LNP-treated groups (Figure 1F). This indicates that the lack of integrin overexpression outside the WOI leads to a loss of RGD-LNP selectivity to the uterus. However, the RGD-LNP also showed a lower luciferase expression in the liver and spleen in mice infused outside the WOI (Figure 1F), suggesting a broader mechanism for reducing systemic exposure with RGD (Figure 1g). Notably, there was >115-fold lower luciferase expression in the cervix compared to the uterus in RGD-LNP-treated mice (Figure 1E), indicating the low mRNA leakage and subsequent uptake by the lower female reproductive tract after intrauterine infusions. The delivery efficiency was determined by calculating the ratio of luciferase expression in the uterus relative to the liver (Figure 1G), and the spleen (Figure 1H) based on data shown in Figures 1E, 1F. There was no statistical significance in the uterus-to-liver or uterus-to-spleen delivery specificity between control- and RGD-LNPs when infused in mice 33 JHU C 17814 PCT 095238 / 00572 outside the WOI (Figure 1G, 1H). However, the RGD-LNP shows much higher uterus-to- liver and uterus-to-spleen delivery ratios compared to the control-LNP when infused during the WOI (Figure 1G, 1H). Further, the RGD-LNP showed a statistically significant higher uterus-to-liver an uterus-to-spleen delivery ratios when infused during the WOI compared to infusion outside the WOI (Figure 1G, 1H). The findings herein provide a novel strategy for the spatiotemporal targeting of mRNA to the uterus during the WOI using RGD-LNPs. Evaluation of intrauterine distribution and penetration of protein expression in RGD-LNP treated mice Motivated by the high luciferase expression in the uterus following intrauterine infusion of RGD-LNP during the WOI, an immunohistochemical analysis was performed to further characterize the tissue distribution and penetration of its protein expression. RGD- LNP encapsulating EGFP mRNA was formulated and infused into the uterus of mice at day 5 p.c. A widespread EGFP signal originating from the luminal epithelium, uterine glands, and stroma of the endometrium was observed 24 hr post-infusion (data not shown). Interestingly, the green signal penetrated the whole endometrial layer with no signal observed in the myometrium of the perimetrium (data not shown). Co-immunostaining was done to confirm the cell types expressing the EGFP in the endometrium. First, a high co-localization between the pan-cytokeratin staining (red) and EGFP expression (green) was observed, indicating the ability of RGD-LNPs to target luminal epithelium (data not shown). Second, the uterus sections with anti-vimentin antibodies were co-stained as a marker of stromal cells. There was no co-localization between the EGFP signal originating from the surface of the endometrium and the stromal cell marker stain, further confirming mRNA delivery to the luminal epithelium, which doesn’t express vimentin (data not shown). Colocalization was observed between the EGFP signal (green) and vimentin (red), demonstrating the ability of RGD-LNP to target endometrial stromal cells (data not shown). An EGFP signal originating from a uterine gland was also observed (data not shown). From a safety viewpoint, no significant EGFP expression was observed in embryonic / decidual tissues at the implantation sites on day 6 p.c (data not shown). The distribution of EGFP in uterine, hepatic, and splenic sections was compared 24 hr post intrauterine infusion of control- and RGD-LNPs. Although control-LNP delivered EGFP mRNA to the endometrial epithelium and stroma similar to RGD-LNP (data not shown), a higher number of cells expressing the EGFP were observed in the liver and spleen compared to RGD-LNPs (data not shown). This was consistent with luciferase expression evaluation (Figure 1E), which showed a lower liver and spleen expression in the RGD-LNP group. 34 JHU C 17814 PCT 095238 / 00572 Engineering RGD-LNPs with reduced off-target exposure following intrauterine infusion In this section, experiments were designed to characterize the RGD-LNP compositions leading to high endometrial targeting and low off-target exposure to the liver and spleen (Figure 2A). To this end, we created a library of 12 LNPs varying in the amount of RGD ligand, the topology of RGD conjugation, and the type of PEG-lipid stabilizer (Figure 2B, 2G). LNPs A-L showed particle size ranging from 94-207 nm, PDI 0.09-0.32, and ζ- potential -1.6 - -16 mV (Table 1). The LNPs were administered by intrauterine infusion in mice at day 5 p.c. to evaluate the local and delivery efficiency and intravenously to determine their systemic tropism (Figure 2A). In Figure 2C, the effect of 3 different densities of C18- RGD (0, 1, and 5 mol%; LNP A-C) were tested. Noteworthy, LNP A and LNP C include the same compositions of control- and RGD-LNPs used in Figures 1A-1H and data not shown, respectively. Increasing the density of C18-RGD increased luciferase expression in the uterus and reduced the expression in the liver and spleen (Figure 2C). Next, whether the site of RGD conjugation on the LNP surface can influence endometrial targeting was tested (Figure 2D). First, to estimate the extent of luciferase expression as a result of integrin-mediated uptake of LNP C by the endometrium at day 5 p.c., an inhibition experiment was performed by pretreating the uterus with vitronectin, a natural ligand for the integrin receptor through the RGD binding site50. The luciferase expression was ~10 times lower in the uterus pre-treated with vitronectin, emphasizing the significant role of integrin-RGD interactions in enhancing uterine mRNA delivery (Figure 2D). Attaching the RGD to a C18 lipid with a 2000 g / mol PEG spacer (C18-PEG2000-RGD) in LNP D (1.5 mol%) and E (5 mol%) showed slightly elevated luciferase expression in the uterus compared to the vitronectin pretreatment baseline (Figure 2D). However, the uterus luciferase expression in LNP C remained 1.8 and 5.8 folds higher than LNP D and LNP E, respectively (Figure 2D). This suggests that attaching high- density targeting ligands directly to the lipid component (LNP C) can enhance endometrial targeting by multivalent interactions. Notably, LNP E with 5 mol% C18-PEG2000-RGD modification resulted in a lower uterus luciferase expression compared to LNP D with 1.5 mol% C18-PEG2000-RGD (Figure 2D). This was concomitant with a reduction in the liver and spleen expression in LNP E compared to D (Figure 2D). This highlights the negative implications of adding excessive amounts of PEG spacers on cellular uptake and trafficking, which can be avoided by using spacer-free conjugation (LNP C vs. LNP E, Figure 2D). Intrauterine infusion of LNP D shows more than 10-fold higher liver expression and around 3-fold higher spleen expression than LNP C (Figure 2D). LNP D also showed approximately 35 JHU C 17814 PCT 095238 / 00572 4-fold higher luciferase expression in the liver and spleen compared to LNP B (Figure 2C, 2D). This indicates that a spacer-free RGD conjugation (LNP B, C) could also reduce systemic expression exposure following intrauterine infusion. Table 1: LNP Formulation Characteristics # LNP ID Size (nm) PDI ζ-potential (mV) 1 LNP A 115 0.09 -8.7 It was proposed that using a short PEG in the PEG-lipid stabilizer component (C18- PEG350) could reduce steric hindrance and enhance RGD exposure on the LNP surface (Figures 1A-1H and data not shown). LNPs were co-formulated with 1.5 mol% C18- PEG2000 and varied the C18-RGD concentration from 0-5 mol% (Figure 2E). There was no enhancement in the luciferase expression in the uterus by increasing the RGD lipid amount (LNPs F-H, Figure 2E). This was opposite to the C18-RGD LNPs co-formulated with 1.5 mol% C18-PEG350 (Figure 2C), which showed higher luciferase expression in the uterus by increasing C18-RGD amounts. This supports the hypothesis that using a short PEG stabilizer component could make the RGD peptides more accessible to interact with the endometrium. Notably, a more than 20-fold reduction in liver and spleen expression was still observed with increasing the C18-RGD density in LNPs F-H (Figure 2E). C18-RGD LNPs was co- formulated with a clinically used PEG-lipid DMG-PEG2000 (C14-PEG2000) at 1.5 mol%. There was a statistically insignificant increase in the uterus expression with the addition of C18-RGD (LNPs I-K; Figure 2F). Additionally, a more than 10-fold reduction in liver and spleen expression was observed with the addition of 5 mol% C18-RGD was seen (Figure 2F). It was also found that 350 g / mol PEG attached to a shorter lipid tail (C14-PEG350) was 36 JHU C 17814 PCT 095238 / 00572 equally effective to C18-PEG350 in delivering mRNA to the uterus (Figure 2G). To get more insights into the multivalency of the RGD-LNPs, the RGD density on the surface of the LNP C was estimated by theoretical calculations (Figures 5A and 5B). In LNP C, functionalized with 5 mol% C18-RGD, the average planar shortest center-to-center distance between adjacent RGD peptides was estimated as 5.2 nm, and the average longest center-to-center distance was estimated as 7.2 nm. LNPs A-L were intravenously injected into mice and evaluated the systemic tissue tropism. Specifically, the luciferase expression in the liver (Figure 3H), the spleen (Figure 6A), and the uterus (Figure 6B). As expected, control LNPs containing 0% RGD (LNP A, F, I) showed high liver luciferase expression (Figure 2H). Increasing the amount of C18-RGD from 0 to 5 mol% reduced liver expression by 100-fold in LNPs A and C, 10-fold in LNPs F and H, and 4-fold in LNPs I and K (Figure 2H). This supports the finding that conjugating the LNPs with RGD peptides directly to the lipid component could reduce the off-target exposure following systemic exposure. It was interesting to observe that even at 5 mol% C18-RGD, LNPs formulated with C14-PEG2000 (LNPs I-K) exhibited high liver expression (Figure 2H), highlighting the liver-tropic characteristic of LNPs formulated with this PEG-lipid. Intriguingly, attaching RGD to the LNP using a PEG spacer (LNP D, 1.5 mol% C18- PEG2000-RGD) still exhibited 2.4- and 14-fold higher liver expression compared to LNPs B and G, respectively (Figure 2H). This highlights the advantage of using a spacer-free RGD to reduce systemic expression. While the intravenous injection of LNPs A-L showed a less predictable effect on spleen expression (Figure 6A), LNP C reduced spleen expression compared to its control (LNP A) by > 2-fold (Figure 6B and Figures 7A and 7B). Intravenous injection of LNPs A-L resulted in negligible luciferase expression in the uterus (Figure 6B), highlighting the need for local infusion to achieve substantial mRNA delivery in the uterus. The % of uterus expression relative to total luciferase expression (uterus, liver, and spleen) after intrauterine infusion for LNPs A-L (Figure.2I) and normalized the liver expression in each LNP to that of LNP C after intravenous injection (Figure.2J). Taken together, LNP C was identified as the composition showing highest LNP expression in the uterus after intrauterine infusion and has the lowest liver tropism after systemic exposure. The luciferase expression in a wider range of mouse organs after the intravenous delivery of LNPs A and C (Figures 7A and 7B). While LNP A showed the highest expression in the liver and spleen, LNP C showed diminished expression in all organs including the liver, spleen, lungs, kidneys, heart, brain, uterus, cervix, and vagina (Figures 7A and 7B). 37 JHU C 17814 PCT 095238 / 00572 The biodistribution of Cy5-labeled mRNA after intravenous injection using IVIS imaging. LNPs A and C were injected at 2 and 6 μg mRNA to enhance the detection sensitivity of the Cy5 signal at the higher dose (data not shown). LNP C exhibited comparable Cy5 signal intensities in the liver (data not shown) and the liver and spleen (data not shown), indicating that RGD-modified LNPs can still accumulate in these organs without inducing functional delivery and subsequent protein expression (Figures 7A and 7B). This was consistent with the LNP ionization measurement by the TNS assay, showing that both LNP A and LNP C have liver-tropic pKa values in the range of 6.2-6.4 (Figure 8). Finally, an approach to minimizing systemic exposure of protein expression of locally administered mRNA-LNPs through direct conjugation of high-density targeting ligands on the LNP surface was introduced (Figure 2K). By employing Bifunctional Ligands via Organized Conjugation (BLOC), endometrial targeting was enhanced via multivalent interactions while simultaneously reducing protein expression in the liver and spleen after systemic exposure (Figure 2K). mRNA-based delivery of a GM-CSF induces localized protein production with minimal plasma exposure To assess the pharmacokinetics of GM-CSF protein expression in mouse tissues, mRNA encoding GM-CSF was encapsulated within LNP C and quantified protein levels using ELISA. First, GM-CSF levels was quantified in the uterus, liver, spleen, and plasma of untreated mice (Figure 3A). GM-CSF tissue levels was measured after intrauterine infusion of fLuc mRNA-loaded LNP C to prevent the LNP upregulating GM-CSF as previously shown31. There was no significant increase in baseline GM-CSF levels in the measured organs after the infusion of fLuc mRNA-loaded LNP C (Figure 3A and 3B). In contrast, a single intrauterine infusion of 4 µg GM-CSF mRNA-loaded LNP C on day 5 p.c. induced substantial GM-CSF production in the infused uterine horn 4 hours post-treatment, which was at least 200-fold higher than that in the liver, spleen, lung, or plasma (Figure 3C). Interestingly, the GM-CSF production in major mouse tissues, including the liver, spleen, and blood plasma, remained comparable to untreated control levels even after the infusion of GM-CSF mRNA (Figure 3A-3C). Minimal GM-CSF expression was observed in the contralateral uterine horn, which was 30-fold lower than the infused uterine horn. In addition, very low GM-CSF production was observed other regions of the female reproductive tract, including the cervix, ovaries, and vagina (Figure 3C). These results underscore the uterus-targeting specificity of mRNA LNP C in producing therapeutic cytokines for endometrial regeneration. 38 JHU C 17814 PCT 095238 / 00572 Protein expression profiles following mRNA- and protein-based were compared. On day 5 p.c., mice received either 4 µg GM-CSF mRNA LNP-C or 3.6 µg recombinant GM- CSF in PBS, with the latter dose matching prior studies in rodents51. To eliminate residual free GM-CSF in the uterine lumen, uteruses were flushed with PBS before tissue processing for ELISA. mRNA-based delivery resulted in sustained protein expression, peaking at 4 hr and returning close to baseline levels by 24 hr post-infusion (Figure 3D). Conversely, recombinant GM-CSF exhibited high initial concentrations in the uterus 1 hr post-infusion but was rapidly eliminated, showing lower levels than mRNA-treated mice at 4 and 8 hr post- infusion by 2.5- and 3.1-fold, respectively (Figure 3D). Further, recombinant GM-CSF induced significantly higher plasma exposure, with an area under the curve (AUC) from 1 to 24 hr that was 60-fold higher than that of mRNA-based infusion (Figure 3E). Specifically, the uterus-to-plasma GM-CSF ratio in mRNA-treated mice was 50-fold higher than in protein- treated mice at 4 hr, and at 8 hr, it remained 3.3-fold higher in the mRNA-treated group (Figure 3F). Taken together, mRNA-based delivery of GM-CSF using LNP C achieved localized and sustained expression in the uterus with minimal systemic exposure. mRNA-based delivery of GM-CSF increases embryo implantation rates in a TE model in mice Finally, the therapeutic utility of the new endometrium targeting strategy was demonstrated in a mouse model of uterine-factor infertility. Thin endometrium (TE) was induced by infusing ethanol into the uterine lumen as previously reported52. Mice were given 7 days after ethanol injury to model the TE (Figure 4A). First, to confirm that GM-CSF can be expressed in the injured endometrium of TE, the TE uterus was treated with GM-CSF mRNA-containing LNP C mice in 2 groups; one group receiving the LNP at day 5 p.c. (during WOI), and the other outside the WOI (Figure 4B). The mice during the WOI showed a high production of GM-CSF in the infused uterine horn and very low plasma exposure levels (Figure 4B). However, the level of GM-CSF expressed in the TE uterus (Figure 4B) was 3-fold lower than that in the healthy uterus (data not shown), which could be attributed to the lower cell viability after ethanol treatment. The injured uterine horn from mice that were not mated showed significantly lower levels of GM-CSF production (Figure 4B), further supporting the role integrins play in enhancing LNP C uptake during the WOI (Figure 1A-1H, 2A-2K and 3A-3F). For efficacy studies, while the right uterine horn was injured with ethanol, the left horn was un-injured and used as an internal control for successful implantation in each mouse (Figure 4A). After 7 days, female mice were mated with males to induce the WOI. On day 2 39 JHU C 17814 PCT 095238 / 00572 p.c., PBS, 4 μg GM-CSF mRNA LNP C, or 3.6 μg recombinant mouse GM-CSF were injected into the right (injured) uterine horn (Figure 4A). Female mice were sacrificed on days 7-9 p.c. to count implantation sites (Figure 4C-4E) and measure endometrial thickness by histopathology (Figure embryo implantation sites compared to the control horn (Figure 4C-4E) and a 110 μm reduction in endometrial thickness (data not shown, Figure 4F). GM- CSF mRNA-loaded LNP C enhanced implantation rates to levels comparable to the non- injured control horn (Figure 4C-4E ). Recombinant GM-CSF showed a 67% reduction in implantation numbers (Figure 4C-4E). This highlights the advantage of using mRNA-based delivery in enhancing the fertility rate in the TE model, presumably by prolonging and sustaining local cytokine expression in the uterus (Figure 4D). It is worth highlighting that although mRNA-LNPs were injected at the peak of WOI (day 5 p.c.) in Figures 1A-1H, 2A- 2K and 3A-3F, treatments were administered on day 2 p.c. in Figure 4C-4E to avoid interfering with the implantation process. When PBS or LNP C were injected in the uterus at day 5 p.c., almost 50% of embryos were aborted (resorbed) upon necropsy on embryonic day 15 (Figure 9). Interestingly, LNP C did not induce a higher abortion rate compared to PBS, suggesting the low toxicity of RGD-modified LNPs on embryogenesis (Figure 9). It has been previously reported that integrin receptor expression increases in mice starting from days 2-3 p.c. and peaks at days 4-546,53,54. This can potentially expand the window in which LNP C can be targeted to the endometrium by utilizing the overexpressed integrins. Discussion Functionalizing nanoparticles with multivalent ligands can maximize particle uptake via active targeting. Many extracellular matrix components contain multiple RGD sequences, which support cell adhesion via multivalent integrin receptor binding. Several adenovirus serotypes have adapted to utilize integrin receptors for cell entry in humans55. Nanomedicine approaches have utilized the RGD sequence to deliver therapeutic cargos to tumor endothelium, placenta, and promote tissue healing56-58. Previously described conjugation approaches have involved attaching the RGD peptides on the terminus of a polymer spacer, typically PEG, to induce interactions with integrins59,60. Here, it was investigated whether direct conjugation of RGD on the lipid component would allow for high-density ligand decoration and promote multivalent receptor interactions instead of conventionally attaching the RGD peptides on a PEG spacer. Notably, RGD binding sites can be reached by peptides that extend only by 1-3 nm from the surface of particles61. The theoretical calculations in the present study showed that LNP C, functionalized with 5 mol% C18-RGD, has a planar shortest center-to-center distance between adjacent RGD peptides of 5.2 nm, and the average 40 JHU C 17814 PCT 095238 / 00572 longest center-to-center distance of 7.2 nm (Figures 5A and 5B). This closely resembles the RGD density on the penton base protein of adenovirus 2, where the shortest distance between immobilized RGDs is 5.7 nm, and the longest distance is 9 nm62. Therefore, the substantial endometrium targeting achieved by LNP C when infused into the mouse uterus during the WOI can be explained by the strong multivalent interactions between overexpressed integrin on the luminal epithelium and the RGD-LNP. It was also observed that attaching RGD to a 2000 g / mol PEG spacer was less effective in inducing protein expression in the uterus compared to the spacer-free approach. It can be reasonably inferred that attaching RGD peptides on the surface of LNP using a 2000 g / mol PEG spacer alters the distance between RGD peptides due to the flexibility of the polymer, reducing the likelihood of multivalent interactions at any given ligand density. Furthermore, PEG can negatively affect cellular uptake and reduce mRNA delivery. It has been previously observed that incorporating a high amount of PEG up to 5 mol% significantly lowers protein expression in vivo63. Therefore, a PEG spacer-free approach can allow for high-density RGD surface functionalization, bypassing the negative implications of excessive PEG on intracellular uptake and trafficking. For the successful formation of mRNA-LNPs, a minimal amount of PEG stabilizer is important for colloidal stability. Conventionally, LNPs are functionalized with a 1.5 mol% PEG-lipid of 2000 g / mol PEG in length. The estimated contour length of a 2000 g / mol PEG in water is 12.7 nm64, which can potentially block the interaction between RGD tethered on the LNP surface and endometrial receptors. Here, a short PEG (350 g / mol) attached to a C18 lipid was used as the PEG-lipid stabilizer component to reduce steric hindrance. While LNPs co-formulated with C18-RGD and C18-PEG350 showed enhancement in luciferase expression in the endometrium, the enhancement was lost when the C18-RGD was co- formulated with a longer PEG (C18-PEG2000), likely due to the steric hindrance. This was further confirmed by measuring the surface charge of LNPs before and after the addition of C18-RGD. In LNPs A-C, the ζ-potential exhibited a significant decrease from -8.7 to -10.8 to -16 mV with the addition of 0, 1.5, and 5 mol% C18-RGD, respectively (Table 1). This could suggest that the anionic C18-RGD is effectively exposed on the LNP surface. However, in LNPs F-H, the ζ-potential change was minimal, decreasing from -1.6 to -4.1 to -4.8 with the addition of 0, 1.5, and 5 mol% C18-RGD, respectively (Table 1). This indicates that the C18- PEG2000 may shield C18-RGD, limiting its surface exposure. It was also found that LNPs co-formulated with C18-RGD and a sheddable PEG-lipid can enhance endometrial targeting slightly. C14-PEG2000 can potentially undergo shedding in the intrauterine cavity similar to 41 JHU C 17814 PCT 095238 / 00572 its desorption upon injection into biological fluids65, exposing the RGD-functionalized surface for interactions with integrins on the endometrium surface. Surface exposure of RGD can be achieved by co-formulating the C18-RGD LNPs with either C18-PEG350 or C14- PEG350. This was consistent with the observation that both LNP C and L have a high negative surface charge (-16 and -15.8 mV for LNP C and LNP L, respectively), indicating the exposure of RGD on the LNP surface. However, a C18-PEG350 seems to endow better LNP size characteristics (LNP C; 135 nm) compared to a C14-PEG350 (LNP L; 207 nm). This is consistent with a previous study showing that incorporating C14-PEG350 into mRNA- LNPs produced larger particle sizes (>250 nm)66. The present study showed that C18- PEG350 can be a suitable PEG-stabilizer component to enhance RGD surface exposure and LNP colloidal stability. mRNA-LNPs can migrate from the local injection site and distribute systemically39, including the liver, presumably by hitchhiking the lymphatic circulation and other unknown mechanisms39,67,68. Extensive research has focused on mitigating the liver exposure of locally delivered mRNA by using carrier-free approaches39, localizing nano-carriers69, or reducing mRNA expression in the liver with microRNA binding sites70. Further, despite the promise of targeting ligands in shifting LNP tropism, liver exposure continues to pose a significant challenge31,71. Here, it was discovered that attaching RGD peptides directly to the lipid component of LNPs reduced their systemic exposure to the liver and spleen. The effect of C18-RGD on reducing off-target exposure was observed irrespective of the delivery window (During WOI vs. outside the WOI), the formulation parameter (LNPs co-formulated with C18-PEG350, C18-PEG2000, C14-PEG2000, or C14-PEG350), and the route of administration (intrauterine vs. intravenous injections). The present data also showed that RGD conjugated onto LNPs using a PEG spacer still exhibited high liver exposure by intravenous and intrauterine administration. PEG spacer may create gaps on the LNP surface, allowing serum lipoproteins to bind more easily, promoting liver uptake. Collectively, we present a new concept for reducing the systemic expression mRNA after local delivery using targeting ligands directly attached to the surface of LNPs. By incorporating Bifunctional Ligands via Organized Conjugation (BLOC), we were able not only to achieve higher endometrium targeting, but also reduced expression in the liver and spleen after systemic exposure. While it was shown that LNP C still accumulates in the liver and spleen, it is believed that the high-density RGD surface functionalization alters its chemistry and attenuates subsequent protein expression in these organs. LNP C shows a 50% reduction in protonation 42 JHU C 17814 PCT 095238 / 00572 capacity in acidic environments (Figure 8), which could lead to inefficient endosomal escape efficiency and lower protein expression in the liver or spleen. However, LNP C demonstrated strong expression in the uterus, indicating that efficient endosomal escape likely occurred in endometrial cells following integrin-receptor-mediated uptake. Previous studies have emphasized the dual role of RGD multivalency in facilitating both cellular internalization and endosomal escape72. Therefore, it can be anticipated that the loss in LNP protonation due to RGD functionalization could reduce its inherent endosomal escape efficiency, but an alternate endosomal escape mechanism in the endometrium could have played a role in achieving cytosolic mRNA delivery. Further investigation is warranted to elucidate the differences in LNP C intracellular trafficking between the uterus and liver. Subcutaneous injections of recombinant CSF cytokines, including those spanning before and after embryo transfer in IVF, were more effective than a single intrauterine infusion before embryo transfer in clinical trials19,24. While effective, excessive GM-CSF can lead to tumor progression, migration, and other immune-related adverse effects73,74. Here, a strategy was provided for sustaining and localizing protein expression in the uterus using a single intrauterine infusion of GM-CSF-mRNA-loaded LNP C. Our novel targeting strategy has the potential to reduce cytokine-induced toxicity and an improved method was provided to isolate local from systemic cytokine effects, which are suitable for studies of localized uterine therapies. Notably, a thin endometrium is not uncommonly encountered in cases where the endometrial cavity is distorted by uterine fibroids, a disease that affects millions of women in the United States75. CSF cytokine treatment was identified as a valuable tool to consider before advising the option of surrogacy76. Taken together, the mRNA-based delivery approach using LNP C proposed herein can potentiate the efficacy and safety of GM-CSF treatment and help many women who lost hope after multiple implantation failures in exhaustive, emotionally taxing, and expensive IVF cycles. While the safety profiles of intrauterine LNPs containing pro-inflammatory ionizable lipids remain to be elucidated, induction of intrauterine inflammation by endometrial scratching or other means was associated with higher embryo implantation rates in humans77-79. Therefore, it can be argued that intrauterine infusion of LNP C can be tolerable in the context of infertility treatment. Finally, the BLOC strategy holds promise in delivering localized mRNA treatments to disease targets that show integrin overexpression, such as endometriosis, inflamed colonic mucosa in inflammatory bowel disease, and solid tumors41-43. References 1 Mareckova, M. et al. Nat Genet 56, 1925-1937 (2024). 43 JHU C 17814 PCT 095238 / 00572 2 Giudice, L. C. & Kao, L. CLancet 364, 1789-1799 (2004). 3 March, C. M. Reprod Biomed Online 23, 63-76 (2011). 4 Lortet-Tieulent, J., J Natl Cancer Inst 110, 354-361 (2018). 5 Murphy, A. R., Campo, H. & Kim, J. J., Nat Rev Endocrinol 18, 727-743 (2022). 6 Xue, Z. et al. 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S., et al., Adv Drug Deliv Rev 99, 28-51 (2016). 50 Park, H. J. et al., Reproduction 153, 351-360 (2017). 51 Wei, W. et al., Mol Hum Reprod 30 (2023). 52 Liu, J. et al., Cytokine 125, 154850 (2020). 53 Sun, Z.-G. Journal of Gynecology and Womens Health 10 (2018). 54 Cai, L., et al., Chinese Science Bulletin 45, 2077-2081 (2000). 55 Ng, Q. K. et al., Mol Ther 17, 828-836 (2009). 56 Kibria, G., et al., Biomaterials 34, 5617-5627 (2013). 57 Katayama, K. et al., Biomaterials 32, 4185-4193 (2011). 58 Clark, A. Y. et al., Nat Commun 11, 114 (2020). 59 Li, Z. et al., Bioorg Med Chem 60, 116704 (2022). 60 Cossu, J., et al., Pharmaceutics 15 (2023). 61 Beer, J. H., et al., Blood 79, 117-128 (1992). 62 Stewart, P. L. et al., EMBO J 16, 1189-1198 (1997). 63 Ryals, R. C. et al., PLoS One 15, e0241006 (2020). 64 Ma, Z. et al., PLoS One 9, e112292 (2014). 65 Mui, B. L. et al., Mol Ther Nucleic Acids 2, e139 (2013). 66 Oberli, M. 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C. o. & Animals, U. o. L. Guide for the care and use of laboratory animals. (US Department of Health and Human Services, Public Health Service, National …, 1986). 46 JHU C 17814 PCT 095238 / 00572

Claims

We claim:

1. A compositions for targeted endometrial delivery of one or more active agents comprising one or more active agents and a population of lipid nanoparticles (LNP), wherein the LNP comprise an ionizable lipid (IL), helper lipid (HL), an integrin targeting ligand / moiety functionalized phospholipid (ITL-PL), and a lipid anchored polymer (PEG-Lipid), optionally wherein the polymer is polyalkylene glycol, wherein each LNP comprises on its surface, one or more targeting ligands (TL) selected from the group consisting of integrins, cadherins, selectins, and immunoglobulins at a density between about 0.5 to about 10 mol% of the total lipid concentration in the LNP.

2. The composition of claim 1, wherein the ITL is at a density between about 0.5 to about 10 mol%, more preferably between 1.5 to about 7 mol% of the total lipid concentration in the LNP, more preferably between about 3 to about 6 mol% of the total lipid concentration in the LNP.

3. The composition of claim 1, comprising a linker between the ITL and the PL, optionally, wherein the linker comprises PEG or is a PEG linker or a linker is an azideAzide / DBCO or thiol / maleimide linker.

4. The composition of any one of claims 1-4, wherein the LNPs comprises an ionizable lipid, a helper lipid, an integrin targeting ligand / moiety functionalized phospholipid, and a lipid anchored hydrophilic polymer, optionally wherein the polymer is selected from the group consisting of a polyalkylene oxide polymer poly(oxazoline), poly(vinyl alcohol), poly(glycerol), poly(N- vinylpyrrolidone), polyaminoacids and poly N-(2- hydroxypropyl)methacrylamide]. poly(oxazoline), poly(vinyl alcohol), poly(glycerol), poly(N- vinylpyrrolidone), polyaminoacids and poly N-(2- hydroxypropyl)methacrylamide].

5. The composition of 4, wherein the polymer-lipid comprises a low molecular weight PEG.

6. The composition of claim 4, comprising a high molecular PEG, preferably where the PEG-Lipid is a PEG-sheddable PEG-Lipid.

7. The composition of claim 5 or 6, comprising a C14-C18 phospholipid, optionally selected from the group of phosphatidylethanolamine lipids comprising C8, C10, C12, C14, C16, C18, C20, 0-3 unsaturated bonds, dilauroylglycerol, dimyristoylglycerol, dipalmitoylglycerol, distearoylgiycerol, dilaurylglycamide, dimyristylglycamide, dipalmitoylglycamide, disterylglycamide, cholesterol, or saturated and unsaturated fatty acids (C8-C20) such as stearic acid and oleic acid. 47 JHU C 17814 PCT 095238 / 005728. The composition of any one of claims 1-7, wherein the ITL-PL comprises a polymer linker.

9. The composition of any of claims 1-7, comprising a ITL-PL, wherein the ITL is directly attached to the PL via a covalent bond.

10. The composition of any one of claims 1-9, comprising a lipid anchored PEG selected from the group consisting of C14-PEG2000: 1,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy-(polyethylene glycol)-2000]; C14-PEG350: 1,2- dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy-(polyethylene glycol)-350]; C14-PEG1000: 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy- (polyethylene glycol)-1000]; C14-PEG3000: 1,2-dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy-(polyethylene glycol)-3000]; C18-PEG2000: 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy-(polyethylene glycol)-2000]; and C18-PEG350: 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy-(polyethylene glycol)-350].

11. The composition of any one of claims 1-10 wherein the ITL is selected from the group consisting of RGD, CRGDCL (SEQ ID NO:1), CRGDCA (SEQ ID NO:2); GACRGDCLGA (SEQ ID NO:3), NGRAHA (SEQ ID NO:4); CVLNGRME (SEQ ID NO:6), RCDVVV (SEQ ID NO:7), SLIDIP (SEQ ID NO:8), and TIRSVD (SEQ ID NO:9).

12. The composition of any one of claims 4-11, further comprising one or more a functional elements conjugated to or otherwise covalently linked, directly (without the presence of an intervening molecule serving as a linker) or indirectly (via an intervening molecule serving as a linker, the LNP..

13. The composition of any one of claims 1-12, comprising one or more active agents are selected from the group consisting of a therapeutic, nutritional, diagnostic, prophylactic compound, or a combination thereof, associated with the LNP.

14. The composition of claim 13, wherein the active agent is selected from the group consisting of a protein, peptide, carbohydrate, polysaccharide, nucleic acid molecule, organic small molecule and / or nucleic acid molecule encoding a therapeutic protein or peptide.

15. The composition of claim 14, comprising a nucleic acid molecule encoding or expressing a therapeutic protein or peptide such as an mRNA.

16. The composition of claim 15, comprising a nucleic acid molecule encoding a growth factor, cytokine, or gene editor.

17. The composition of claim 14, wherein the active agent is selected from the group consisting of GM-CSF, hGH, Cas9, and Cre recombinase. 48 JHU C 17814 PCT 095238 / 0057218. A method of treating a subject in need thereof comprising administering an effective amount of the composition of any one of claims 1-17 to a female subject during the subject’s implantation window.

19. The method of claim 18, wherein the subject is a human subject, wherein the composition is administered at about day 20-24 of the human subject’s menstrual cycle, or 6- 10 days after ovulation.

20. The method of claim 18 or 19 wherein the subject has been diagnosed with endometriosis.

21. The method of claim 18-20, wherein the subject has been diagnosed with endometrial cancer.

22. The method of claim 18, wherein the composition is administered prior to an infertility treatment.

23. The method of any one of claims 18-22, wherein the composition is administered intrauterine or intravaginally. 49 JHU C 17814 PCT 095238 / 00572

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