Placenta-targeted nanoparticles or conjugates and methods of use thereof

Abstract

Provided herein are compositions, methods, and particles that include a peptide that targets placental chondroitin sulfate A (placental CSA), wherein the amino acid sequence of the polypeptide is as set forth in SEQ ID NO: 1 (EDVKDINFDTKEKFLAGLIVSFHEGKC).

Description

PLACENTA-TARGETED NANOPARTICLES OR CONJUGATES AND METHODS OF USE THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Serial No. 63 / 668,025, filed July 5, 2024, the entire contents of which are incorporated herein by reference.TECHNICAL FIELD OF THE INVENTION

[0002] The present invention relates in general to the field of targeting nano and microparticles, and more particularly, to novel placenta-targeted particles or conjugates and methods of use thereof.STATEMENT OF FEDERALLY FUNDED RESEARCH

[0003] This invention was made with government support under AA031684 awarded by the National Institutes of Health. The government has certain rights in this invention.INCORPORATION-BY-REFERENCE OF MATERIALS FILED ON COMPACT DISC

[0004] The present application includes a Sequence Listing which has been submitted in XML format via EFS-Web and is hereby incorporated by reference in its entirety. Said XML copy, created on , 2025, is named .xml and is , bytes in size.BACKGROUND OF THE INVENTION

[0005] Without limiting the scope of the invention, its background is described in connection with targeting the placenta.

[0006] One such placental targeting agent is taught in Patent Publication No. WO2019061648 Al, filed by Xiujun, et al., entitled “Polypeptide targeting placental chondroitin sulfate A, targeted delivery system, and preparation method and application thereof’. These applicants are said to teach a polypeptide targeting placental chondroitin sulfate A, a targeted delivery system targeting placental chondroitin sulfate A, and a preparation method, in which the polypeptide and targeted delivery system provided by the present invention are highly targeted to tissue expressing placental chondroitin sulfate A.

[0007] Another such placental targeting agent is taught in U.S. Patent Publication No. 20180298098, filed by Zocca, entitled “Immunotherapeutic targeting of placental-like chondroitin sulfate using chimeric antigen receptors (CARS) and immunotherapeutic targeting of cancer using CARS with split-protein binding systems”. This application is said to teach polypeptidescomprising conditionally active chimeric antigen receptor (CAR) and a split-protein binding system, a VAR2CSA polypeptide, or any other targeting agent, such as relates to polypeptides comprising VAR2CSA polypeptides or other targeting agent, and a split-protein binding system.

[0008] Another such placental targeting agent is taught in U.S. Patent Publication No. 20060094672, filed by Pasqualini, et al., entitled “Compositions and Methods of Use of Targeting Peptides Against Placenta And Adipose Tissues”. This application is said to teach that placentatargeting peptides may be used for targeted delivery of therapeutic agents to placenta and / or fetus. And in some embodiments, these applicants teach receptors identified by binding to placentatargeting peptides may be used to screen compounds for potential teratogenicity.

[0009] Despite these advances, a need remains for improved targeting of the placenta, including, a need to improve the delivery of active agents using particles targeted to the placenta with no off- target adverse effects on the fetus.SUMMARY OF THE INVENTION

[0010] As embodied and broadly described herein, an aspect of the present disclosure relates to a peptide that targets placental chondroitin sulfate A (placental CSA), wherein an amino acid sequence of the peptide is as set forth in SEQ ID NO: 1 (EDVKDINFDTKEKFLAGLIVSFHEGKC). As embodied and broadly described herein, an aspect of the present disclosure relates to a targeted delivery system that targets placenta-like chondroitin sulfate A, wherein the targeted delivery system comprises the peptide of SEQ ID NO: 1 (EDVKDINFDTKEKFLAGLIVSFHEGKC). In one aspect, the peptide is not EDVKDINFDTKEKFLAGCLIVSFHEGK (SEQ ID NO: 2). In another aspect, the peptide is conjugated to phospholipid and the conjugated peptide-phospholipid is incorporated into a particle. In another aspect, the particle further comprises one or more of the following components: a hydrophobic core, a surfactant, a phospholipid, a hydrophobic polymer, an amphiphilic macromolecule grafted to the polypeptide, or an amphiphilic macromolecule interspersed in a lipid monolayer or bilayer. In another aspect, the particle is spherical and has a diameter of nanometers or micrometers. In another aspect, the particle further comprises one or more active agents or biomarkers for placental targeting. In another aspect, the one or more active agents are selected for treating a placental disease or a disease that traverse a placental barrier, or not traverse the placental barrier, selected from active agents to treat Fetal Alcohol Spectrum Disorder (FASD), preeclampsia, gestational diabetes, intrauterine growth restriction, target vaccines or prophylactic agents to enhance fetal immunity, target placental tumors or abnormalities, placenta associated syndromes, placental abruption, placenta previa, small for gestational age, preterm or still birth;normalize redox homeostasis, glutathione synthesis, or activate a transsulfuration pathway selected from chlorogenic acid and caffeic acid, and derivatives thereof; NFATc4 / NFAT3 transcriptional regulators; regulators of cysteine transport (EAAC1 (SLC1A1)) and cystine transport (Xc- (SLC7A11)); activation of Nrf2 (nuclear factor E2-related factor 2) selected from resveratrol and KI696; compounds supplying cysteine for glutathione synthesis selected from N- acetyl cysteine and derivatives thereof; proangiogenic factors selected from VEGF (VEGF-A mRNA), Netrin-1 and Notchl signaling, activators of glutathione peroxidase; and selenium. In another aspect, the one or more biomarkers are selected from: a contrast agent, a fluorescence tracer, a photothermal conversion reagent, a molecule to study placental physiology or drug transport during pregnancy, or combinations thereof. In another aspect, the amphiphilic macromolecule is a polyethylene glycol derivatized phospholipid, the polyethylene glycol derivatized phospholipid being passed from polyethylene glycol and its derivatives. In another aspect, the amphiphilic macromolecule is a polyethylene glycol -derivatized phospholipid, and the polyethylene glycol -derivatized phospholipid is passed through a polyethylene glycol and a derivative thereof, and one or more viscous or viscosity enhancing molecules selected from at least one of polyvinyl alcohol, glucose, hyaluronic acid, or gelatin. In another aspect, the targeted delivery system further comprises at least one of: one or more proteins, one or more carbohydrates, one or more lipids, or one or more small molecules. In another aspect, the targeted delivery system further comprises one or more excipients or carriers selected from: pharmaceutical carriers, inorganic nanomaterials, diluents, adhesives, buffers, or salts. In another aspect, the targeted delivery system delivers a second active agent or a pregnancy drug for treating gestational diabetes, treating pregnancy syndrome, treating intrauterine growth retardation, anti-epilepsy, anti-inflammatory, premature delivery, prevents premature rupture of membranes, a polypeptide drug, and or a gene therapy.

[0011] As embodied and broadly described herein, an aspect of the present disclosure relates to a method of preparing a targeted delivery system, comprising the steps of: conjugating a peptide EDVKDINFDTKEKFLAGLIVSFHEGKC of SEQ ID NO: 1 to a phospholipid and the conjugated peptide-phospholipid is incorporated into a particle; mixing the conjugated peptide with one or more phospholipids to form a monolayer or bilayer; adding one or more surfactants, carriers, active agents or payloads into the mixture; and ultrasonically treating the mixture to form active agent loaded particles. In one aspect, the peptide is not EDVKDINFDTKEKFLAGCLIVSFHEGK (SEQ ID NO: 2). In another aspect, the peptide is attached or conjugated to the one or more phospholipid and the conjugated peptide-phospholipid is incorporated into a particle. In another aspect, the particle further comprises one or more of thefollowing components: a hydrophobic core, a surfactant, a phospholipid, a hydrophobic polymer, an amphiphilic macromolecule grafted to the peptide, or an amphiphilic macromolecule interspersed in a lipid monolayer or bilayer. In another aspect, the particle is spherical and has a diameter of nanometers or micrometers. In another aspect, the particle further comprises one or more active agents or biomarkers for placental targeting. In another aspect, the one or more active agents are selected for treating a placental disease or a disease that traverse a placental barrier, or not traverse the placental barrier, selected from active agents to treat Fetal Alcohol Spectrum Disorder (FASD), preeclampsia, gestational diabetes, intrauterine growth restriction, target vaccines or prophylactic agents to enhance fetal immunity, target placental tumors or abnormalities, placenta associated syndromes, placental abruption, placenta previa, small for gestational age, preterm or still birth; normalize redox homeostasis, glutathione synthesis, or activate a transsulfuration pathway selected from chlorogenic acid and caffeic acid, and derivatives thereof; NFATc4 / NFAT3 transcriptional regulators; regulators of cysteine transport (EAAC1 (SLC1A1)) and cystine transport (Xc- (SLC7A11)); activation of Nrf2 (nuclear factor E2-related factor 2) selected from resveratrol and KI696; compounds supplying cysteine for glutathione synthesis selected from N-acetyl cysteine and derivatives thereof; proangiogenic factors selected from VEGF (VEGF-A mRNA), Netrin-1 and Notch 1 signaling, activators of glutathione peroxidase; and selenium. In another aspect, the particle further comprises one or more biomarkers selected from at least one of: a contrast agent, a fluorescence tracer, a photothermal conversion reagent, a molecule to study placental physiology or drug transport during pregnancy, or combinations thereof. In another aspect, the amphiphilic macromolecule is a polyethylene glycol derivatized phospholipid, the polyethylene glycol derivatized phospholipid being passed from polyethylene glycol and its derivatives. In another aspect, the amphiphilic macromolecule is a polyethylene glycol-derivatized phospholipid, and the polyethylene glycol-derivatized phospholipid is passed through a polyethylene glycol and a derivative thereof, and one or more viscous or viscosity enhancing molecules selected from at least one of polyvinyl alcohol, glucose, hyaluronic acid, or gelatin. In another aspect, the targeted delivery system further comprises at least one of: one or more proteins, one or more carbohydrates, one or more lipids, or one or more small molecules. In another aspect, the targeted delivery system further comprises one or more excipients or carriers selected from: pharmaceutical carriers, inorganic nanomaterials, diluents, adhesives, buffers, or salts. In another aspect, the targeted delivery system delivers a pregnancy drug for treating gestational diabetes, treating pregnancy syndrome, treating intrauterine growth retardation, anti-epilepsy, anti-inflammatory, premature delivery, prevents premature rupture of membranes, a polypeptide drug, and or a gene therapy.

[0012] As embodied and broadly described herein, an aspect of the present disclosure relates to a method of treating a pregnancy disease or targeting the placenta comprising: identifying a patient in need of treatment for the pregnancy disease or in need of targeting the placenta; and providing the patient with a therapeutically effective amount of a pharmaceutical composition comprising the peptide of SEQ ID NO: 1 (EDVKDINFDTKEKFLAGLIVSFHEGKC). In one aspect, the pregnancy disease or targeting a placenta is selected from Fetal Alcohol Spectrum Disorder (FASD), preeclampsia, gestational diabetes, intrauterine growth restriction, target vaccines or prophylactic agents to enhance fetal immunity, target placental tumors or abnormalities, placenta associated syndromes, placental abruption, placenta previa, small for gestational age, preterm or still birth, or combinations thereof. In another aspect, the method further comprises one or more active agents are selected for treating a placental disease or a disease that traverse a placental barrier, or not traverse the placental barrier, selected from active agents to treat Fetal Alcohol Spectrum Disorder (FASD), preeclampsia, gestational diabetes, intrauterine growth restriction, target vaccines or prophylactic agents to enhance fetal immunity, target placental tumors or abnormalities, placenta associated syndromes, placental abruption, placenta previa, small for gestational age, preterm or still birth; normalize redox homeostasis, glutathione synthesis, or activate a transsulfuration pathway selected from chlorogenic acid and caffeic acid, and derivatives thereof; NFATc4 / NFAT3 transcriptional regulators; regulators of cysteine transport (EAAC1 (SLC1A1)) and cystine transport (Xc- (SLC7A11)); activation of Nrf2 (nuclear factor E2-related factor 2) selected from resveratrol and KI696; compounds supplying cysteine for glutathione synthesis selected from N-acetyl cysteine and derivatives thereof; proangiogenic factors selected from VEGF (VEGF-A mRNA), Netrin-1 and Notch 1 signaling, activators of glutathione peroxidase; and selenium.

[0013] As embodied and broadly described herein, an aspect of the present disclosure relates to a pharmaceutical preparation for treating a pregnancy disease or targeting a placenta, wherein the pharmaceutical preparation for treating a pregnancy disease or targeting the placenta comprises the peptide of SEQ ID NO: 1 (EDVKDINFDTKEKFLAGLIVSFHEGKC) or a targeted delivery system comprising SEQ ID NO: 1 (EDVKDINFDTKEKFLAGLIVSFHEGKC). In one aspect, the peptide is used to target active agents to treat Fetal Alcohol Spectrum Disorder (FASD), preeclampsia, gestational diabetes, intrauterine growth restriction, target vaccines or prophylactic agents to enhance fetal immunity, target placental tumors or abnormalities, placenta associated syndromes, placental abruption, placenta previa, small for gestational age, preterm or still birth, or combinations thereof. In another aspect, the pharmaceutical preparation further comprises one or more active agents are selected for treating a placental disease or a disease that traverse aplacental barrier, or not traverse the placental barrier, selected from active agents to treat Fetal Alcohol Spectrum Disorder (FASD), preeclampsia, gestational diabetes, intrauterine growth restriction, target vaccines or prophylactic agents to enhance fetal immunity, target placental tumors or abnormalities, placenta associated syndromes, placental abruption, placenta previa, small for gestational age, preterm or still birth; normalize redox homeostasis, glutathione synthesis, or activate a transsulfuration pathway selected from chlorogenic acid and caffeic acid, and derivatives thereof; NFATc4 / NFAT3 transcriptional regulators; regulators of cysteine transport (EAAC1 (SLC1 Al)) and cystine transport (Xc- (SLC7A11)); activation of Nrf2 (nuclear factor E2 -related factor 2) selected from resveratrol and KI696; compounds supplying cysteine for glutathione synthesis selected from N-acetyl cysteine and derivatives thereof; proangiogenic factors selected from VEGF (VEGF-A mRNA), Netrin-1 and Notch 1 signaling, activators of glutathione peroxidase; and selenium.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] For a more complete understanding of the features and advantages of the present disclosure, reference is now made to the detailed description of the disclosure along with the accompanying figures and in which:

[0015] FIG. 1 shows an overview of damage on a mechanistic level caused by ethanol, which enables targeted rescue. In utero ethanol (EtOH) exposure impairs the Placental-Cortex Axis which can be defined as “placental changes that guide fetal brain development”. Recent studies have shown that disruption of this Axis by alcohol elicits FASD [documented in humans and in rodent models]. Prior studies from the inventor’s lab have shown that EtOH causes oxidative damage in placenta and fetal brain by impairing redox homeostasis. This dysregulation is caused by impaired maintenance of glutathione (GSH) homeostasis. The inventors have shown that this damage can be prevented by manipulations that prevent the loss GSH.

[0016] FIG. 2 is an illustration which shows the CSA-targeting peptide EDVKDINFDTKEKFLAGLIVSFHEGKC (SEQ ID NO: 1) conjugated to DSPE-PEG5k- mal eimide. Placental chondroitin sulfate A (plCSA)-targeting peptide is conjugated to 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-5000] (DSPE-PEG5k) and incorporated on the NP surface for targeted delivery of NP -loaded drugs to adipose stromal cells. NPs are synthesized using soy phosphatidylcholine (PC), a-tocopherol acetate, Kolliphor® HS15 (surfactant), DSPE-PEG5k and water. Drug-loaded NPs are small, stable, biocompatible, and easily synthesized. For trophoblast targeting, the inventors have incorporated the linear placental chondroitin sulfate A (plCSA)-targeting peptide(EDVKDINFDTKEKFLAGLIVSFHEGKC, SEQ ID NO: 1) on the NP surface. As illustrated, this CSA-targeting peptide is conjugated to DSPE-PEG5k-maleimide, which replaced DSPE- PEG5k used in non-targeted drug-loaded NPs. Maleimide on DSPE-PEG5k reacts and couples with the thiol group of cysteine (C) on the CSA-targeting peptide to form DSPE-PEG5k-Peptide conjugate (FIG. 2). Conjugation has been confirmed by a MALDI / TOF Mass Spectrometer. DSPE and PC anchor onto the NP membrane by burying their two fatty acid tails inside the NP hydrophobic core that consists of a-tocopherol acetate, compound of choice or the PEG5k and conjugated PEK5k-peptide sections protrude towards the outside aqueous environment (FIG. 2). PEG5k maintains integrity and stability of NPs. The targeting peptide, conjugated to the top of PEG5k in opposite of DSPE, protrudes toward external environment with high binding affinity to trophoblasts, at their CSA receptor.

[0017] FIG. 3 shows a comparison of expression of nanoparticles using LI (EDVKDINFDTKEKFLAGCLIVSFHEGKC, SEQ ID NO: 2), and L2 (EDVKDINFDTKEKFLAGLIVSFHEGKC, SEQ ID NO: 1) as the targeting peptide, using expression of Nrf2 in HTR8 cells.

[0018] FIG. 4 shows a comparison of expression of nanoparticles using LI (EDVKDINFDTKEKFLAGCLIVSFHEGKC, SEQ ID NO: 2), and L2 (EDVKDINFDTKEKFLAGLIVSFHEGKC, SEQ ID NO: 1) as the targeting peptide, using expression of Nrf2 in Rat Placenta.

[0019] FIGS. 5 A and 5B show in vitro studies illustrating targeting success with human-derived syncytiotrophoblast cells. FIG. 5A shows the differentiation of human-derived BeWo cells to chondroitin sulfate A -containing syncytiotrophoblast (STB) cells after a 48-hour Forskolin treatment and targeted drug delivery by nanoparticle to STB cells. Fig. 5B shows when these human-derived syncytiotrophoblast were exposed to resveratrol loaded nanoparticles (Targeted) for 24 hours, the drug was successfully delivered. The cellular Trans-resveratrol content in STB cells treated with EDVKDINFDTKEKFLAGLIVSFHEGKC, L2 (SEQ ID NO: 1) targeted NP compared to non-targeted (void) was ~2.7-fold higher.

[0020] FIGS. 6A to 6C show that ethanol (EtOH) decreased expression of Cth, NFAT3, Nrf2 and the two transporters xCT (cystine) / SLC7Al 1 and EAAC1 (cysteine) / SLClAl in sy ncy ti otrophobl asts / S TB .

[0021] FIGS. 7 A and 7B show that L2 (EDVKDINFDTKEKFLAGLIVSFHEGKC, SEQ ID NO: 1) targeted NPs (Targeted) loaded with / ra / rs-Resveratrol increased the target gene expression in human derived differentiated BeWo cells. 7ra / / .s-resveratrol activated the transcription factorNFE2L2, eliciting increases in the three intervention targets, the Cys / CySS transporters CS7 / ’7.47 / EAAC I and SLC7A17 / xCT) and Cth, the rate limiting enzyme in the transsulfuration pathway.

[0022] FIG. 8 shows the placental / fetal nanoparticle localization in hours post material injection, and the in vivo administration of the nanoparticles is specific to placenta with none reaching the fetus. Specific targeting of the placenta using plCSA NP is illustrated using maternally administered (tail-vein) nanoparticles (NP) where none of the attached fluorescent probes on the R-containing nanoparticles enters the fetus, only the placenta (FIG. 8). Presence of NPs in placenta is shown at 4, 6, and 24 hours post NP injection. Subsequent studies illustrated that the transresveratrol was specifically delivered to the placenta (none detected in the fetus) and elicited an increase in targeted gene expression (FIG. 10).

[0023] FIG. 9 shows the Trans-resveratrol content in the rat placenta treated with EDVKDINFDTKEKFLAGLIVSFHEGKC, L2 (SEQ ID NO: 1) targeted NP (Targeted) compared to non-targeted (Void) was ~3.4-fold higher.

[0024] FIGS. 10 show that resveratrol is delivered to the placenta in sufficient concentrations to up-regulate Nrf2 and Cth. Pregnant dams were injected with resveratrol containing nanoparticles via tail vein. Resveratrol is delivered (“Targeted” bars) (FIG. 10 Nrf2). FIG. 10 Cth shows that the resveratrol containing nanoparticles upregulated the master transcription factor, Nrf2, and increased Cth mRNA expression, a control point for the transsulfuration pathway. This, reactivates cysteine synthesis (rescuing Glutathione-related redox homeostasis) and potentially, FES- mediated angiogenesis. Impaired angiogenesis has been shown in ethanol-exposed placenta and has been mechanistically connected to multiple other compromised pregnancy outcomes.

[0025] FIGS. 11A and 11B show that specific placental delivery of resveratrol can significantly normalize the ethanol-induced decrease of mRNA expression of the vital cystine transporter (Slc7all / xCT) in both rat placenta (FIG. 11 A) and fetal brain (FIG. 1 IB). There is an extensive literature connecting this transporter to enabling control of cellular redox homeostasis. In vivo ethanol (EtOH) exposure is from gestation days (GD) 11 to 20 (second trimester equivalent). The NPs containing Resveratrol (NP) were injected i.v. (tail vein) on GD18 and the placenta and corresponding fetal brain were harvested on GD20. Targeted Resveratrol-NP treatment significantly rescued expression of the Cystine transporter (Slc7al 7 / xCT), vital to control of redox homeostasis, in both placenta and fetal brain.

[0026] FIGS. 12A and 12B show specific placental delivery of resveratrol prevents the Ethanol (EtOH)-induced oxidative damage that the inventors and others have shown elicits placentadysfunction and fetal neuron apoptotic death. Most importantly, this is compelling evidence that the nanoparticle delivery system can rescue the fetal brain by specifically targeting the placenta. In vivo ethanol (EtOH) exposure is from gestation days (GD) 11 to 20 (second trimester equivalent). The NPs containing Resveratrol (NP) were injected i.v. (tail vein) on GDI 8 and the placenta and corresponding fetal brain were harvested on GD20. 4-hydroxynonenal (4-HNE) is a highly toxic lipid peroxidation end product which forms stable protein adducts. Ethanol-induced 4-HNE production has been shown to play a mechanistic role in fetal brain proapoptotic cell death responses to ethanol. Measuring 4-HNE adducts is a widely accepted measure of oxidative damage.DETAILED DESCRIPTION OF THE INVENTION

[0027] While the making and using of various aspects of the present disclosure are discussed in detail below, it should be appreciated that the present disclosure provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific aspects discussed herein are merely illustrative of specific ways to make and use the disclosure and do not delimit the scope of the disclosure.

[0028] To facilitate the understanding of this disclosure, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present disclosure. Terms such as “a”, “an” and “the” are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific aspects of the disclosure, but their usage does not delimit the disclosure, except as outlined in the claims.

[0029] Pregnancy -related complications and disorders, such as Fetal Alcohol Spectrum Disorder (FASD), preeclampsia, gestational diabetes, and intrauterine growth restriction, and other placenta-associated syndromes like placental abruption, placental previa, small for gestational age (SGA) preterm or still birth, often necessitate the targeted delivery of medications to the placenta. Traditional drug delivery methods may not provide the desired efficacy and they expose the fetus to these drugs which risks damaging fetal development. The inventors have developed a novel nanotechnology-based drug delivery system that utilizes nanoparticles to specifically target and deliver therapeutic agents to the placenta, specifically to the placental chondroitin sulfate A receptor in the apical membrane of the placental syncytiotrophoblast. This innovation involves the encapsulation of therapeutic compounds within biocompatible nanoparticles engineered to have a high affinity for placental tissue. By leveraging the unique biological characteristics of the placenta, these nanoparticles will not cross the maternal-fetal barrier, ensuring the precise delivery of drugs to the placental target site without exposing the developing fetus to undue risks.

[0030] Alcohol consumption during pregnancy is a well-documented risk factor for neurodevelopmental disorders, including fetal alcohol syndrome and the more prevalent fetal alcohol spectrum disorder. These conditions cause lifelong physical, behavioral, and cognitive impairments. However, no effective clinical interventions are currently available. Neuroplacentology is the study of how the placenta influences fetal brain development through the placenta-brain axis. Neuroplacentology explores how placental dysfunction contributes to neurodevelopmental disorders, highlighting the placenta’s critical role in fetal brain health.

[0031] The present invention uses placenta-targeted nanoparticles to direct active agent(s), which has the potential to revolutionize drug delivery during pregnancy, to enhance the health and wellbeing of both mother and fetus.

[0032] Current research approaches cannot deliver agents to the placenta precisely. The present invention demonstrates the following advantages: (1) precise and controlled delivery to trophoblast and syncytiotrophoblast cells in the placenta; (2) minimized risk to the developing fetus due to specific targeting; (3) improved therapeutic outcomes and prenatal diagnostics; and / or (4) contributes tools and reagents to advance placental research using the novel targeting agent to deliver contrast agents to the placenta.

[0033] Presently, no clinical interventions are available to specifically mitigate placental dysfunction related to, e.g., Fetal Alcohol Spectrum Disorder (FASD), preeclampsia, gestational diabetes, and intrauterine growth restriction. The present invention specifically targets drug delivery to the placenta with no fetal exposure (FIG. 8). This unique component is the use of a novel placenta-targeting peptide EDVKDINFDTKEKFLAGLIVSFHEGKC (SEQ ID NO: 1) to the nanoparticle surface. In summary, advantages of the present invention are: (1) precise and controlled drug delivery to the placenta; (2) minimized systemic exposure, reduced fetal risk, and improved fetal development; (3) improved therapeutic outcomes for pregnancy-related disorders; and (4) enhanced tools for understanding of placental biology and drug transport mechanisms.

[0034] The present inventors successfully incorporated the trophoblast / syncytiotrophoblast- targeting peptide (EDVKDINFDTKEKFLAGLIVSFHEGKC) (SEQ ID NO: 1) on the nanoparticle (NP) surface. Conjugation has been confirmed by a MALDI / TOF Mass Spectrometer.

[0035] FIG. 1 shows an overview of damage on a mechanistic level caused by ethanol, which enables targeted rescue. In Utero ethanol (EtOH) exposure impairs the Placental-Cortex Axis which can be defined as “placental changes that guide fetal brain development”. Recent studies have shown that disruption of this Axis by alcohol elicits FASD [documented in humans and inrodent models]. Prior studies from the inventor’s lab have shown that EtOH causes oxidative damage in placenta and fetal brain by impairing redox homeostasis. This dysregulation is caused by impaired maintenance of glutathione (GSH) homeostasis. The inventors have shown that this damage can be prevented by manipulations that prevent the loss GSH.

[0036] As an example, the inventors generated nanoparticles (NPs) containing / ra / rs-Resveratrol (R) with the surface bound ligand. Functional Testing: (1) Targeting is specific: Specific targeting of the placenta using plCSA NP, and (2) the drug encapsulated in the NPs is functionally delivered to the placenta, and the packaged / ra / / .s-Resveratrol becomes active in the placenta, activating Nrf2 transcription factor, thereby increasing gene expression of two of the key intervention targets, the cystine transporter xCT and Cth, the rate limiting enzyme in transsulfuration pathway (FIGS. 5B, 9, 7A, 7B, 3, 4) (FIGS. 6A-6C, 7A, 7B, 8, 9). The present inventors have previously shown that up-regulating these two targets can prevent oxidative damage to the placenta and fetal brain components.

[0037] Previously, a peptide having the amino acid sequence EDVKDINFDTKEKFLAGCLIVSFHEGKC (SEQ ID NO: 2) (see WO2019061648A1), was used to target the placenta.

[0038] The present inventors sought to improve the targeting of the placenta and to enhance the delivery of active agent(s) using particles that used a novel targeting peptide with enhanced delivery of targeting and active agents. As shown herein, the two peptides are compared and EDVKDINFDTKEKFLAGLIVSFHEGKC (SEQ ID NO: 1) showed superior efficacy for targeting and delivery of active agent(s) (FIGS. 3 and 4) (FIGS. 8 and 9).

[0039] Using the present invention, cell-type-specific gene expression changes can be targeted in the placenta during second-trimester ethanol (EtOH) exposure, which contributes to impaired brain development, leading to postnatal behavioral deficits. EtOH is a well-established teratogen. Furthermore, using the present invention to deliver active agents that actively target these cell- type-specific gene expression changes, second-trimester ethanol (EtOH) exposure can be mitigated using nanoparticle-based targeted drug delivery to placental syncytiotrophoblast cells.

[0040] The nanoparticle drug delivery system disclosed herein, which specifically targets placenta, can be used to optimize placental function, thereby normalizing fetal brain development altered by EtOH. A transcriptomic approach can be used to identify new molecular targets for rescue of placental function to support fetal brain development. In addition, computational analyses can also be used to rescue the teratogenic effects of EtOH in fetal brain by using a new specific placenta targeted drug delivery approach.

[0041] For example, the nanoparticle drug delivery system disclosed herein can be used in rescue strategies that prevent EtOH- induced impairments using specific nanoparticle mediated drug delivery to placental syncytiotrophoblast cells to prevent fetal exposure to maternally- administrated medications that could elicit off-target adverse responses e.g. impaired angiogenesis, synaptic terminal growth, and Wnt signaling.

[0042] The present inventors have shown that placenta dysfunction caused by EtOH, shown both in utero in rats and in vitro, decreases nuclear Nrf2, a master regulator of the cellular oxidative stress responses and diminishes its transactivation ability along with dysregulation of proliferation indices, PCNA, CYCLIN-D1, and p21. It is also known that EtOH impairs placental amino acid uptake and placental oxidative stress (OS) is associated with human pregnancy complications including preeclampsia and fetal growth restriction. Impaired glutathione (GSH)-related redox homeostasis is the key source of the damaging oxidative stress.

[0043] The nanoparticle drug delivery system disclosed herein can be used with an in uteroHn vivo model. EtOH exposure system generates a rapid onset of OS. The in vivo rat model uses a vapor chamber-based prenatal alcohol exposure model (La Jolla Alcohol Research Inc., La Jolla, CA, USA). The vapor chamber maintains a reliable exposure level and allows minimal handling / restraint stress. Rats (EtOH and Control) remain in their plastic cages during treatment, thereby minimizing stress related to environmental changes. Timed pregnant rats are received on gestation day (GD) 4 (GD4) and EtOH treatment begins on GDI 1 allowing time to acclimate to the new environment. Timed pregnant Sprague Dawley rats receive an intermittent 95% EtOH vapor daily with a 6 h ON / 18 h OFF in a vapor chamber for periods between GD 11-20. Blood alcohol level is maintained at 160-185 mg / dL, monitored through GDs. This is an exposure of a second-trimester equivalent in human brain development, in the absence of mature astrocytes and their GSH homeostasis support. Rationale for this timeline is that GDI 1-20 is an active phase of fetal CNS development including neurogenesis, vasculogenesis, angiogenesis, proliferation, migration, and differentiation. Only by gestation days (GD)12 in rats, does the placenta acquire a structure that has a layer of trophoblast giant cells, spongiotrophoblast, and the inner labyrinth (equivalent to human villi and where nutrient transfer occurs), which until term undergoes expansion leading to placental growth. Sex-related factors are highly relevant and males and females will be assessed by sex typing using multiplex PCR analysis for kdm5c gene (x chromosome) and SRY gene (y chromosome).

[0044] Transcriptomics, bioinformatics. A discovery-based approach can be used to identify celltype specific gene expression changes in placenta exposed to EtOH by means of RNA Sequencing (RNA-Seq) and several bioinformatic approaches, including differential gene expression (DEG),functional group over-representation analysis and cell type over-representation analysis. There are 36 cell types identified in placenta and the effects of prenatal EtOH exposure on different cell types is determined. To validate the molecular findings from the transcriptomic approach, using the nanoparticle drug delivery system of the present invention, it is possible to target cell typespecific gene networks using a targeted pharmacological screening approach to identify rescue targets capable of reversing molecular changes and restoring placental function.

[0045] Materials and Methods.

[0046] Nanoparticle (NP) Generation and Targeting Strategy.

[0047] Drug-loaded nanoparticles (NPs) will be synthesized using soy phosphatidylcholine (PC), a- tocopherol acetate, Kolliphor® HS15 (surfactant), DSPE-PEG5k, and water, following established protocols (12-14). These NPs are biocompatible, stable, and easily manufactured. To enable trophoblast-specific targeting, the inventors will incorporate a placental chondroitin sulfate A (plCSA)-binding peptide (EDVKDINFDTKEKFLAGLIVSFHEGKC (SEQ ID NO:1)) on the NP surface. This peptide is conjugated to DSPE- PEG5k-maleimide, replacing unconjugated DSPE-PEG5k used in non-targeted NPs. The maleimide group reacts with the cysteine thiol of the peptide, forming a stable DSPE-PEG5k-peptide conjugate, confirmed by MALDI-TOF mass spectrometry. The amphiphilic structure of DSPE and PC allows them to anchor into the hydrophobic NP core, which contains a-tocopherol acetate and / or drug cargo. PEG5k provides steric stability, while the peptide extends outward for specific CSA receptor binding on trophoblasts.

[0048] Formulation, Optimization, and Characterization.

[0049] NP composition has been optimized for targeting efficiency, drug loading, and stability. Key parameters like particle size, zeta potential, poly dispersity, encapsulation efficiency, and drug loading are measured using a Malvern Zetasizer, TEM, and Agilent 1290 Infinity II UHPLC system. Encapsulation occurs during sonication in hot water, embedding the compound within the hydrophobic core. This ensures thermal stability and prevents premature drug release prior to placental uptake.

[0050] NP Administration in Pregnant Rats.

[0051] Drug-loaded NPs will be administered via tail vein injection to pregnant rats (FIG. 3). Both ethanol-exposed and control dams will receive identical NP treatments, initiated at optimized time points before ethanol exposure. Based on prior studies (FIGS. 3 and 4), the inventors will verify NP distribution and circulation time under both conditions to refine dosing schedules. Control groups will receive NPs with or without the encapsulated drug.

[0052] Assessment of Target Specificity.

[0053] To evaluate biodistribution, DiR-labeled NPs will be visualized using the IVIS® Lumina XR system at 1, 2-, 4-, 6-, and 24-hours post-injection. Rats will be euthanized at each time point to harvest maternal organs, placentas, and fetuses for NP quantification. Distribution of the encapsulated compound will be determined using established analytical methods (14).

[0054] Rescue Strategy Experiments:

[0055] Experiment 1 : Placental Rescue N a.Nrf2 Activation Using Resveratrol.

[0056] The inventors will evaluate the therapeutic potential of resveratrol-loaded NPs to restore redox homeostasis disrupted by prenatal ethanol exposure. Markers of oxidative stress and redox imbalance — including HNE-protein adducts, malondialdehyde, DNA fragmentation, PARP-1 expression, apoptotic markers, and Nrf2 levels — will be quantified in the placenta and matched fetal cerebral cortex (4-7, 9).

[0057] Experiment 2: Rescue via Novel Druggable Targets Identified by Transcriptomic and Computational Analyses Based on the hypothesis that transcriptomic dysregulation underlies ethanol-induced placental dysfunction, the inventors will use computational drug- repurposing strategies to identify compounds capable of normalizing placental gene networks. Candidate drugs will be tested in vivo for their efficacy in rescuing ethanol -induced molecular and developmental abnormalities.

[0058] Transcriptomic and computational approaches to identify novel druggable targets.

[0059] The primary transcriptomic approach will be 3 ’-Tag RNA sequencing (RNA-Seq), which will enable identification of differentially expressed genes (DEGs), molecular pathways, and functional cellular states associated with placental dysfunction and its impaired support of fetal brain development. For example, using the present invention it is possible to focus will be placed on pathways related to redox homeostasis. Sex-specific gene expression patterns in placentas and fetal brains from male and female offspring can also be examined, with appropriate controls.

[0060] The nanoparticle drug delivery system disclosed herein can be used to determine the effect of prenatal alcohol exposure (PAE) on global gene expression in placenta and prenatal brain. Bulk RNA-Seq will identify differentially expressed genes (DEGs) in each tissue, followed by functional group and cell type over-representation analyses.

[0061] The nanoparticle drug delivery system disclosed herein can be used to determine the effects of prenatal alcohol exposure (PAE) on placenta-brain communication / interactions. Gene network analysis (WGCNA) is used to identify PAE-responsive cell type - specific gene networksin each tissue, followed by correlational analysis identifying gene networks corelating between placenta and brain.

[0062] Using the present invention, (1) specific mechanisms underlying PAE-induced detrimental effects on brain development are determined, and (2) biomarkers and therapeutic targets for fetal alcohol spectrum disorders (FASD) are validated.

[0063] Experimental Design: Bulk RN A- Sequencing (Bulk RNA-Seq): Bulk RNA-Seq will be performed on placentas and its corresponding fetal brain tissues from control and alcohol -exposed animals. Established protocols are used to analyze the transcriptomes. Placental tissues and its corresponding fetal brain are collected from three timed-pregnant rats per group (Control and EtOH exposed). To investigate sex differences in the placenta and fetal brain in PAE, placentas are sequenced, as are corresponding fetal brain from four male and four female fetuses per group (Control vs. EtOH-exposed), resulting in a total of 48 samples. Each placenta is hemi-dissected: one half for RNA isolation for 3 ’ -Tag sequencing and validation via qPCR, while the other half is used for protein validation via Western blot. Similarly for fetal brain the frontal cortex is dissected and used for total RNA as well as protein isolation. The isolated RNA is sequenced on the Illumina NovaSeq 6000 platform at approximately 3-5 million reads per sample (2x100 bp). Data is normalized, and differentially expressed genes (DEGs) as well as overrepresented functional groups will be identified.

[0064] RNA-Seq Analysis Comparisons: Data is analyzed with two-way ANOVA, with EtOH and sex as two between-subject factors. Interaction effects (EtOH x sex) is evaluated.

[0065] A dosage unit of the placental targeting particles of the present invention may be used to deliver a single compound or mixtures thereof with other compounds. The compound may be mixed together, form ionic, or even covalent bonds with the particles or parts thereof. The placental targeting particles of the present invention may be administered in oral, intravenous (bolus or infusion), intraperitoneal, subcutaneous, or intramuscular form, all using dosage forms well known to those of ordinary skill in the pharmaceutical arts. Depending on the particular location or method of delivery, different dosage forms, e.g., tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions may be used to provide the placental targeting particles of the present invention to a patient in need of therapy that includes the placental targeting particles. The placental targeting particles may also be administered as any one of known salt forms.

[0066] The placental-targeting particles are typically administered in admixture with suitable pharmaceutical salts, buffers, diluents, extenders, excipients and / or carriers (collectively referredto herein as a pharmaceutically acceptable carrier or carrier materials) selected based on the intended form of administration and as consistent with conventional pharmaceutical practices. Depending on the best location for administration, the placental targeting particles may be formulated to provide, e.g., maximum and / or consistent dosing for the particular form for oral, rectal, topical, intravenous injection or parenteral administration. While the placental targeting particles may be administered alone, it will generally be provided in a stable salt form mixed with a pharmaceutically acceptable carrier. The carrier may be solid or liquid, depending on the type and / or location of administration selected.

[0067] Techniques and compositions for making useful dosage forms using the present invention to carry one or more active agents, contrast agents, etc., are described in one or more of the following references: Anderson, Philip O.; Knoben, James E.; Troutman, William G, eds., Handbook of Clinical Drug Data, Tenth Edition, McGraw-Hill, 2002; Pratt and Taylor, eds., Principles of Drug Action, Third Edition, Churchill Livingston, New York, 1990; Katzung, ed., Basic and Clinical Pharmacology, Ninth Edition, McGraw Hill, 2007; Goodman and Gilman, eds., The Pharmacological Basis of Therapeutics, Tenth Edition, McGraw Hill, 2001; Remington’s Pharmaceutical Sciences, 20th Ed., Lippincott Williams & Wilkins., 2000, and updates thereto; Martindale, The Extra Pharmacopoeia, Thirty- Second Edition (The Pharmaceutical Press, London, 1999); all of which are incorporated by reference, and the like, relevant portions incorporated herein by reference.

[0068] For example, the placental targeting particles may be included in a tablet. Tablets may contain, e.g., suitable binders, lubricants, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents and / or melting agents. For example, oral administration may be in a dosage unit form of a tablet, gelcap, caplet or capsule, the active drug component being combined with a non-toxic, pharmaceutically acceptable, inert carrier such as lactose, gelatin, agar, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol, mixtures thereof, and the like. Suitable binders for use with the present invention include: starch, gelatin, natural sugars (e.g., glucose or beta-lactose), com sweeteners, natural and synthetic gums (e.g., acacia, tragacanth or sodium alginate), carboxymethylcellulose, polyethylene glycol, waxes, and the like. Lubricants for use with the invention may include: sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, mixtures thereof, and the like. Disintegrators may include, e.g., starch, methyl cellulose, agar, bentonite, xanthan gum, mixtures thereof, and the like.

[0069] Placental targeting particles may be administered in the form of liposome delivery systems, e.g., small unilamellar vesicles, large unilamellar vesicles, and multilam ellar vesicles, whethercharged or uncharged. Liposomes may include one or more: phospholipids (e.g., cholesterol), stearylamine and / or phosphatidylcholines, mixtures thereof, and the like.

[0070] Placental targeting particles may also be coupled to one or more soluble, biodegradable, bioacceptable polymers as drug carriers or as a prodrug. Such polymers may include: polyvinylpyrrolidone, pyran copolymer, polyhydroxylpropylmethacrylamide-phenol, polyhydroxyethylasparta-midephenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues, mixtures thereof, and the like. Furthermore, the placental targeting particles may be coupled one or more biodegradable polymers to achieve controlled release of the placental targeting particles, biodegradable polymers for use with the present invention include: polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates, and crosslinked or amphipathic block copolymers of hydrogels, mixtures thereof, and the like.

[0071] In one embodiment, gelatin capsules (gelcaps) may include the placental targeting particles and powdered carriers, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Like diluents may be used to make compressed tablets. Both tablets and capsules may be manufactured as immediate-release, mixed-release or sustained-release formulations to provide for a range of release of medication over a period of minutes to hours. Compressed tablets may be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere. An enteric coating may be used to provide selective disintegration in, e.g., the gastrointestinal tract.

[0072] For oral administration in a liquid dosage form, the oral drug components may be combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Examples of suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and / or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents, mixtures thereof, and the like.

[0073] Liquid dosage forms of the placental targeting particles for oral administration may also include coloring and flavoring agents that increase patient acceptance and therefore compliance with a dosing regimen. In general, water, a suitable oil, saline, aqueous dextrose (e.g., glucose, lactose and related sugar solutions) and glycols (e.g., propylene glycol or polyethylene glycols) may be used as suitable carriers for parenteral solutions. Solutions for parenteral administrationinclude generally, a water-soluble salt of the active ingredient, suitable stabilizing agents, and if necessary, buffering salts. Antioxidizing agents such as sodium bisulfite, sodium sulfite and / or ascorbic acid, either alone or in combination, are suitable stabilizing agents. Citric acid and its salts and sodium EDTA may also be included to increase stability. In addition, parenteral solutions may include pharmaceutically acceptable preservatives, e.g., benzalkonium chloride, methyl- or propyl-paraben, and / or chlorobutanol. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field, relevant portions incorporated herein by reference.

[0074] For dermal and transdermal delivery, the placental targeting particles may be delivered using lotions, creams, oils, elixirs, serums, transdermal skin patches and the like, as are well known to those of ordinary skill in that art. Parenteral and intravenous forms may also include pharmaceutically acceptable salts and / or minerals and other materials to make them compatible with the type of injection or delivery system chosen, e.g., a buffered, isotonic solution.

[0075] Capsules. Capsules with the placental targeting particles may be prepared by filling standard two-piece hard gelatin capsules each with 10 to 500 milligrams of powdered active ingredient, 5 to 150 milligrams of lactose, 5 to 50 milligrams of cellulose and 6 milligrams magnesium stearate.

[0076] Soft Gelatin Capsules. A mixture of placental targeting particles is dissolved in a digestible oil such as soybean oil, cottonseed oil or olive oil. The active ingredient is prepared and injected by using a positive displacement pump into gelatin to form soft gelatin capsules containing, e.g., 1, 5, 10, 15, 20, 35, 30, 35, 40, 45, 50, 60, 70, 75, 80, 90, 100, 200, 300, 400, 500, 600, 700, 750, 800, 900, or 1,000 micro- or milli-grams of the active ingredient. The capsules are washed and dried.

[0077] Tablets. A large number of tablets are prepared by conventional procedures so that the dosage unit was 100-500 milligrams of active ingredient, 0.2 milligrams of colloidal silicon dioxide, 5 milligrams of magnesium stearate, 50-275 milligrams of microcrystalline cellulose, 11 milligrams of starch and 98.8 milligrams of lactose. Appropriate coatings may be applied to increase palatability or delay absorption.

[0078] To provide an effervescent tablet appropriate amount of, e.g., monosodium citrate and sodium bicarbonate, are blended together and then roller compacted, in the absence of water, to form flakes that are then crushed to give granulates. The granulates are then combined with the active ingredient, drug and / or salt thereof, conventional beading or filling agents and, optionally, sweeteners, flavors and lubricants.

[0079] Injectable solution. A parenteral composition suitable for administration by injection is prepared by stirring 1% to 80% by weight of active ingredient in deionized water and mixed with, e.g., up to 10% by volume propylene glycol and water. In some aspects, the weight percent of the active agent is 1, 2, 3, 4, 5, 6, 7, 8, 910, 15, 20, 25, 30, 40, 50, 60, 70, 75, or 80%. The solution is made isotonic with sodium chloride and sterilized using, e.g., ultrafiltration.

[0080] Suspension. An aqueous suspension is prepared for oral administration so that each 5 ml contain 100 mg of finely divided active ingredient, 200 mg of sodium carboxymethyl cellulose, 5 mg of sodium benzoate, 1.0 g of sorbitol solution, U.S.P., and 0.025 ml of vanillin.

[0081] For mini-tablets, the active ingredient is compressed into a hardness in the range 6 to 12 Kp. The hardness of the final tablets is influenced by the linear roller compaction strength used in preparing the granulates, which are influenced by the particle size of, e.g., the monosodium hydrogen carbonate and sodium hydrogen carbonate. For smaller particle sizes, a linear roller compaction strength of about 15 to 20 KN / cm may be used.

[0082] Kits. The present invention also includes pharmaceutical kits useful, for example, for the treatment of cancer, which comprise one or more containers containing a pharmaceutical composition comprising a therapeutically effective amount of placental targeting particles. Such kits may further include, if desired, one or more of various conventional pharmaceutical kit components, such as, for example, containers with one or more pharmaceutically acceptable carriers, additional containers, etc., as will be readily apparent to those skilled in the art. Printed instructions, either as inserts or as labels, indicating quantities of the components to be administered, guidelines for administration, and / or guidelines for mixing the components, may also be included in the kit. It should be understood that although the specified materials and conditions are important in practicing the invention, unspecified materials and conditions are not excluded so long as they do not prevent the benefits of the invention from being realized.

[0083] Examples of suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and / or suspensions reconstituted from non- effervescent granules and effervescent preparations reconstituted from effervescent granules. Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents. Oral dosage forms optionally contain flavorings and coloring agents. Parenteral and intravenous forms may also include minerals and other materials to make them compatible with the type of injection or delivery system chosen.

[0084] As used herein, the term “chewable” refers to semi-soft, palatable and stable chewable treat without addition of water. It should be appreciated to the skilled artisan that a chewable composition will be stable and palatable, fast disintegrating, semi-soft medicated chewable tablets (treats) by extrusion without the addition of extraneous water. A soft chewable tablet does not harden on storage and are resistant to microbial contamination. A semi-soft chewable contain a blend of any one or more of binders, flavors, palatability enhancers, humectants, disintegrating agents, non-aqueous solvents, and diluents that are plasticized with liquid plasticizers, such as glycols and polyols to make them ductile and extrudable. The chewable can be made by extrusion, e.g., including fats or lipids as plasticizers and binding agents, is manufactured in the absence of added water, uses plasticizers to replace water in extrudable matrices, contains humectants to maintain the extrudable chew in a pliant and soft state during its shelf life, or any combination thereof. The chewable form may be provided in conjunction with one or more flavorings and / or taste masking agents that improve the taste of the formulation greater than 10, 20, 30, 40, 50, 60, 70, 80, or 90%. The chewable can include the active agent and the ion exchange resin to enhance taste masking.

[0085] For topical administration, the placental targeting particles can be incorporated into creams, ointments, gels, transdermal patches and the like. The composition can also be incorporated into medical dressings, for example wound dressings e.g. woven (e.g. fabric) dressings or non-woven dressings (e.g. gels or dressings with a gel component). The use of alginate polymers in dressings is known, and such dressings, or indeed any dressings, may further incorporate the alginate oligomers of the invention.

[0086] As used herein, the term “active ingredient(s),” “pharmaceutical ingredient(s),” “active agent(s)” and “bioactive agent(s)” are defined as drugs and / or pharmaceutically active ingredients. The placental targeting particles of the present invention may be used to envelop, encapsulate, attach, bind or otherwise be used to affect the storage, stability, longevity and / or release of any of the following drugs as the pharmaceutically active agent in a composition.

[0087] Non-limiting examples of active agents include, but are not limited to, antibiotics, analgesics, vaccines, anticonvulsants; antidiabetic agents, antifungal agents, antineoplastic agents, antioxidants, antiparkinsonian agents, antirheumatic agents, appetite suppressants, biological response modifiers, cardiovascular agents, central nervous system stimulants, contraceptive agents, dietary supplements, vitamins, minerals, lipids, saccharides, metals, amino acids (and precursors), nucleic acids and precursors, contrast agents, diagnostic agents, dopamine receptor agonists, erectile dysfunction agents, fertility agents, gastrointestinal agents, hormones, immunomodulators, antihypercalcemia agents, mast cell stabilizers, muscle relaxants, nutritionalagents, ophthalmic agents, osteoporosis agents, psychotherapeutic agents, parasympathomimetic agents, parasympatholytic agents, respiratory agents, sedative hypnotic agents, skin and mucous membrane agents, smoking cessation agents, steroids, sympatholytic agents, urinary tract agents, uterine relaxants, vaginal agents, vasodilator, anti -hypertensive, hyperthyroids, antihyperthyroids, anti-asthmatics and vertigo agents. In certain embodiments, the one or more therapeutic compounds are water-soluble, poorly water-soluble drug or a drug with a low, medium or high melting point. The therapeutic compounds may be provided with or without a stabilizing salt or salts.

[0088] One or more of the following active agents may be combined with one or more carriers and the present invention (which may itself be the carrier):

[0089] Analgesic anti-inflammatory agents such as, acetaminophen, aspirin, salicylic acid, methyl salicylate, choline salicylate, glycol salicylate, 1 -menthol, camphor, mefenamic acid, fluphenamic acid, indomethacin, diclofenac, alclofenac, ibuprofen, ketoprofen, naproxene, pranoprofen, fenoprofen, sulindac, fenbufen, clidanac, flurbiprofen, indoprofen, protizidic acid, fentiazac, tolmetin, tiaprofenic acid, bendazac, bufexamac, piroxicam, phenylbutazone, oxyphenbutazone, clofezone, pentazocine, mepirizole, and the like.

[0090] Drugs having an action on the central nervous system, for example sedatives, hypnotics, antianxiety agents, analgesics and anesthetics, such as, chloral, buprenorphine, naloxone, haloperidol, fluphenazine, pentobarbital, phenobarbital, secobarbital, amobarbital, cydobarbital, codeine, lidocaine, tetracaine, dyclonine, dibucaine, cocaine, procaine, mepivacaine, bupivacaine, etidocaine, prilocaine, benzocaine, fentanyl, nicotine, and the like.

[0091] Antihistaminics or antiallergic agents such as, diphenhydramine, dimenhydrinate, perphenazine, triprolidine, pyrilamine, chlorcyclizine, promethazine, carbinoxamine, tripelennamine, brompheniramine, hydroxyzine, cyclizine, meclizine, clorprenaline, terfenadine, chlorpheniramine, and the like. Anti-allergenics such as, antazoline, methapyrilene, chlorpheniramine, pyrilamine, pheniramine, and the like.

[0092] Decongestants such as phenylephrine, ephedrine, naphazoline, tetrahydrozoline, and the like.

[0093] Antipyretics such as aspirin, salicylamide, non-steroidal anti-inflammatory agents, and the like. Antimigraine agents such as, dihydroergotamine, pizotyline, and the like.

[0094] Acetonide anti-inflammatory agents, such as hydrocortisone, cortisone, dexamethasone, fluocinolone, triamcinolone, medrysone, prednisolone, flurandrenolide, prednisone, halcinonide, methylprednisolone, fludrocortisone, corticosterone, paramethasone, betamethasone, ibuprofen,naproxen, fenoprofen, fenbufen, flurbiprofen, indoprofen, ketoprofen, suprofen, indomethacin, piroxicam, aspirin, salicylic acid, diflunisal, methyl salicylate, phenylbutazone, sulindac, mefenamic acid, meclofenamate sodium, tolmetin, and the like.

[0095] Steroids such as, androgenic steroids, such as, testosterone, methyltestosterone, fluoxymesterone, estrogens such as, conjugated estrogens, esterified estrogens, estropipate, 17-|3 estradiol, 17-|3 estradiol valerate, equilin, mestranol, estrone, estriol, 17[3 ethinyl estradiol, di ethylstilbestrol, progestational agents, such as, progesterone, 19-norprogesterone, norethindrone, norethindrone acetate, melengestrol, chlormadinone, ethisterone, medroxyprogesterone acetate, hydroxyprogesterone caproate, ethynodiol diacetate, norethynodrel, 17-a hydroxyprogesterone, dydrogesterone, dimethi sterone, ethinylestrenol, norgestrel, demegestone, promegestone, megestrol acetate, and the like.

[0096] Respiratory agents such as, theophilline and [^-adrenergic agonists, such as, albuterol, terbutaline, metaproterenol, ritodrine, carbuterol, fenoterol, quinterenol, rimiterol, solmefamol, soterenol, tetroquinol, and the like.

[0097] Sympathomimetics such as, dopamine, norepinephrine, phenylpropanolamine, phenylephrine, pseudoephedrine, amphetamine, propylhexedrine, arecoline, and the like.

[0098] Local anesthetics such as, benzocaine, procaine, dibucaine, lidocaine, and the like.

[0099] Antimicrobial agents including antibacterial agents, antifungal agents, antimycotic agents and antiviral agents; tetracyclines such as, oxytetracycline, penicillins, such as, ampicillin, cephalosporins such as, cefalotin, aminoglycosides, such as, kanamycin, macrolides such as, erythromycin, chloramphenicol, iodides, nitrofrantoin, nystatin, amphotericin, fradiomycin, sulfonamides, purrolnitrin, clotrimazole, miconazole chloramphenicol, sulfacetamide, sulfamethazine, sulfadiazine, sulfamerazine, sulfamethizole and sulfisoxazole; antivirals, including idoxuridine; clarithromycin; and other anti-infectives including nitrofurazone, and the like.

[0100] Antihypertensive agents such as, clonidine, a-methyldopa, reserpine, syrosingopine, rescinnamine, cinnarizine, hydrazine, prazosin, and the like. Antihypertensive diuretics such as, chlorothiazide, hydrochlorothrazide, bendoflumethazide, trichlormethiazide, furosemide, tripamide, methylclothiazide, penfluzide, hydrothiazide, spironolactone, metolazone, and the like. Cardiotonics such as, digitalis, ubidecarenone, dopamine, and the like. Coronary vasodilators such as, organic nitrates such as, nitroglycerine, isosorbitol dinitrate, erythritol tetranitrate, and pentaerythritol tetranitrate, dipyridamole, dilazep, trapidil, trimetazidine, and the like. Vasoconstrictors such as, dihydroergotamine, dihydroergotoxine, and the like. [3-blockers oranti arrhythmic agents such as, timolol pindolol, propranolol, and the like. Humoral agents such as, the prostaglandins, natural and synthetic, for example PGEi, PGE201, and PGF201, and the PGEi analog misoprostol. Antispasmodics such as, atropine, methantheline, papaverine, cinnamedrine, methscopolamine, and the like.

[0101] Calcium antagonists and other circulatory organ agents, such as, aptopril, diltiazem, nifedipine, nicardipine, verapamil, bencyclane, ifenprodil tartarate, molsidomine, clonidine, prazosin, and the like. Anti -convul sants such as, nitrazepam, meprobamate, phenytoin, and the like. Agents for dizziness such as, isoprenaline, betahistine, scopolamine, and the like. Tranquilizers such as, reserprine, chlorpromazine, and antianxiety benzodiazepines such as, alprazolam, chlordiazepoxide, clorazeptate, halazepam, oxazepam, prazepam, clonazepam, flurazepam, triazolam, lorazepam, diazepam, and the like.

[0102] Antipsychotics such as, phenothiazines including thiopropazate, chlorpromazine, triflupromazine, mesoridazine, piperracetazine, thioridazine, acetophenazine, fluphenazine, perphenazine, trifluoperazine, and other major tranquilizers such as, chlorprathixene, thiothixene, haloperidol, bromperidol, loxapine, and molindone, as well as, those agents used at lower doses in the treatment of nausea, vomiting, and the like.

[0103] Muscle relaxants such as, tolperisone, baclofen, dantrolene sodium, cyclobenzaprine.

[0104] Drugs for Parkinson's disease, spasticity, and acute muscle spasms such as levodopa, carbidopa, amantadine, apomorphine, bromocriptine, selegiline (deprenyl), trihexyphenidyl hydrochloride, benztropine mesylate, procyclidine hydrochloride, baclofen, diazepam, dantrolene, and the like. Respiratory agents such as, codeine, ephedrine, isoproterenol, dextromethorphan, orciprenaline, ipratropium bromide, cromglycic acid, and the like. Non-steroidal hormones or antihormones such as, corticotropin, oxytocin, vasopressin, salivary hormone, thyroid hormone, adrenal hormone, kallikrein, insulin, oxendolone, and the like.

[0105] Vitamins such as, vitamins A, B, C, D, E and K and derivatives thereof, calciferols, mecobalamin, and the like for dermatologically use. Enzymes such as, lysozyme, urokinaze, and the like. Herb medicines or crude extracts such as, Aloe vera, and the like.

[0106] Agents that are capable of normalizing redox homeostasis and supporting glutathione synthesis including compounds activating the transsulfuration pathway such as chlorogenic acid and caffeic acid (and derivatives thereof) and NFATc4 / NFAT3 transcriptional regulators, regulators of cysteine transport (EAAC1 (SLC1A1)) and cystine transport (Xc- (SLC7A11)), activation of Nrf2 (nuclear factor E2 -related factor 2) such as resveratrol and KI696, compounds supplying cysteine for glutathione synthesis such as N-acetyl cysteine and derivatives thereof,proangiogenic factors such as VEGF (VEGF-A mRNA), Netrin-1 and Notch 1 signaling optimization, activators of glutathione peroxidase such as selenium.

[0107] Antitumor agents such as, 5 -fluorouracil and derivatives thereof, krestin, picibanil, ancitabine, cytarabine, and the like. Anti-estrogen or anti-hormone agents such as, tamoxifen or human chorionic gonadotropin, and the like. Miotics such as pilocarpine, and the like.

[0108] Cholinergic agonists such as, choline, acetylcholine, methacholine, carbachol, bethanechol, pilocarpine, muscarine, arecoline, and the like. Antimuscarinic or muscarinic cholinergic blocking agents such as, atropine, scopolamine, homatropine, methscopolamine, homatropine methylbromide, methantheline, cyclopentolate, tropicamide, propantheline, anisotropine, dicyclomine, eucatropine, and the like.

[0109] Mydriatics such as, atropine, cyclopentolate, homatropine, scopolamine, tropicamide, eucatropine, hydroxyamphetamine, and the like. Psychic energizers such as 3-(2- aminopropy)indole, 3-(2-aminobutyl)indole, and the like.

[0110] Antidepressant drugs such as, isocarboxazid, phenelzine, tranylcypromine, imipramine, amitriptyline, trimipramine, doxepin, desipramine, nortriptyline, protriptyline, amoxapine, maprotiline, trazodone, and the like.

[0111] Anti-diabetics such as, insulin, and anticancer drugs such as, tamoxifen, methotrexate, and the like.

[0112] Anorectic drugs such as, dextroamphetamine, methamphetamine, phenylpropanolamine, fenfluramine, diethylpropion, mazindol, phentermine, and the like.

[0113] Anti-malarial agents such as, the 4-aminoquinolines, alphaaminoquinolines, atovaquone- proguanil, chloroquine, mefloquine, primaquine, pyrimethamine, quinine sulfate, tafenoquine, and the like.

[0114] Anti-parasitic agents, e.g., for the treatment of Toxoplasma gondii, such as, pyrimethamine and sulfadiazine, trimethoprim-sulfamethoxazole (TMP-SMX), clindamycin, atovaquone, spiramycin, dapsone, azithromycin, minocycline, and rifabutin.

[0115] Anti-microbials, e.g., for the treatment of Listeria monocytogenes, such as, ampicillin, penicillin G, gentamicin, trimethoprim-sulfamethoxazole, vancomycin, erythromycin, and fluoroquinolones.

[0116] Anti -ulcerative agents such as, misoprostol, omeprazole, enprostil, and the like.

[0117] Antiulcer agents such as, allantoin, al di oxa, al cl oxa, N-methylscopolamine methylsuflate, and the like. Antidiabetics such as insulin, and the like.

[0118] For use with vaccines, one or more antigens, such as, natural, heat-killer, inactivated, synthetic, peptides and even T cell epitopes (e.g., GADE, DAGE, MAGE, etc.) and the like.

[0119] The drugs mentioned above may be used in combination as required. Moreover, the above drugs may be used either in the free form or, if capable of forming salts, in the form of a salt with a suitable acid or base. If the drugs have a carboxyl group, their esters may be employed.

[0120] The acid mentioned above may be an organic acid, for example, methanesulfonic acid, lactic acid, tartaric acid, fumaric acid, maleic acid, acetic acid, or an inorganic acid, for example, hydrochloric acid, hydrobromic acid, phosphoric acid or sulfuric acid. The base may be an organic base, for example, ammonia, triethylamine, or an inorganic base, for example, sodium hydroxide or potassium hydroxide. The esters mentioned above may be alkyl esters, aryl esters, aralkyl esters, and the like.

[0121] When a drug different than an anesthetic agent is used the solvent selected is one in that the drug is soluble. In generally the polyhydric alcohol may be used as a solvent for a wide variety of drugs. Other useful solvents are those known to solubilize the drugs in question.

[0122] Nanoparticle Design: The placenta-targeted nanoparticles were designed to have a size range, surface charge, and surface functionalization that enhance their placental affinity while minimizing non-specific interactions. Surface modifications may include ligands that bind specifically to placental receptors or other molecules overexpressed in placental tissue.

[0123] The inventors successfully incorporated the trophoblast / syncytiotrophoblast-targeting peptide EDVKDINFDTKEKFLAGLIVSFHEGKC (SEQ ID NO: 1) on the nanoparticle surface. For comparison, the inventors also incorporated a comparison peptide: EDVKDINFDTKEKFLAGCLIVSFHEGK (SEQ ID NO: 2). As shown herein, peptide EDVKDINFDTKEKFLAGLIVSFHEGKC (SEQ ID NO: 1) has superior binding and targeting of the placenta when incorporated on the surface of a particle, specifically, a nanoparticle (FIGS. 3 and 4) (FIG. 8).

[0124] Therapeutic Encapsulation: The nanoparticles can encapsulate a wide-range of therapeutic agents, including small molecules, proteins, nucleic acids, or other biologically active compounds. This encapsulation ensures the stability and controlled release of the therapeutic payload.

[0125] Sustained and Controlled Release: The nanoparticles are designed for sustained and controlled release of the therapeutic agents within the placenta. This ensures a sustained and optimal therapeutic effect while minimizing systemic and fetal exposure and potential side effects.

[0126] Increased delivery and therapeutic efficacy and decreased side effects and toxicity: The targeted delivery can enhance the therapeutic efficacy and reduce side effects and toxicity.

[0127] The present invention can be used for the following. As a treatment of pregnancy-related complications such as FASD, preeclampsia, gestational diabetes, and intrauterine growth restriction. For delivery of vaccines or prophylactic agents to enhance fetal immunity. It can also be used for targeted therapy for placental tumors or abnormalities. The peptide and particles can also be used with investigational tools for studying placental physiology and drug transport during pregnancy.

[0128] The present invention was used for precise and controlled drug delivery to the placenta. It was also found to minimize systemic exposure, reduce fetal risk, and improve fetal development. The particles of the present invention can be used to improve therapeutic outcomes for pregnancy- related disorders, and to enhance understanding of placental biology and drug transport mechanisms.

[0129] Nanoparticles were synthesized using soy phosphatidylcholine (PC), a-tocopherol acetate, Kolliphor® HS15 (surfactant), l,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)- PEG5k or DSPE-PEG2k and water. Drug-loaded nanoparticles are small, stable, biocompatible, and easily synthesized. For trophoblast targeting, the inventors incorporated the linear placentatargeting peptide EDVKDINFDTKEKFLAGLIVSFHEGKC (SEQ ID NO: 1) on the nanoparticle surface. The targeting peptide was conjugated to DSPE-PEG5k-maleimide / DSPE-PEG2k- maleimide, and DSPE-PEG5k / DSPE-PEG2k was used in non-targeted drug-loaded nanoparticles. Maleimide on DSPE-PEG5k / DSPE-PEG2k reacts and couples with the thiol group of cysteine (C) on the targeting peptide to form DSPE-PEG5k-Peptide conjugate. Conjugation has been confirmed by a MALDI / TOF Mass Spectrometer. As a comparison, the peptide of the prior art, EDVKDINFDTKEKFLAGCLIVSFHEGK (SEQ ID NO: 2), was also conjugated as described herein above. For the synthesis of nanoparticles, the lipids which include other forms of vitamin E, or triglyceride, or other lipids can be used to replace a-tocopherol acetate. Other surfactants to replace Kolliphor® HS15 may include Tween20, Tween80, etc.

[0130] As illustrated in FIG. 2, the CSA-targeting peptide EDVKDINFDTKEKFLAGLIVSFHEGKC (SEQ ID NO: 1) or SEQ ID NO: 1 peptide were conjugated to DSPE-PEG5k-maleimide / DSPE-PEG2k-maleimide, which replaced DSPE- PEG5k / DSPE-PEG2k used in non-targeted drug-loaded NPs. Maleimide on DSPE-PEG5k reacts and couples with the thiol group of cysteine (C) on the CSA-targeting peptide to form DSPE- PEG5k-Peptide / DSPE-PEG2k-Peptide conjugate (FIG. 2). Conjugation has been confirmed by a MALDI / TOF Mass Spectrometer. DSPE and PC anchor onto the NP membrane by burying theirtwo fatty acid tails inside the NP hydrophobic core that consists of a-tocopherol acetate, compound of choice or the PEG5k / PEG2k and conjugated PEK5k-peptide / PEG2k-peptide sections protrude towards the outside aqueous environment (FIG. 2). PEG5k / PEG2k maintains integrity and stability of NPs. The targeting peptide, conjugated to the top of PEG5k / PEG2k in opposite of DSPE, protrudes toward external environment with high binding affinity to trophoblasts, at their CSA receptor.

[0131] NP Administration: Pregnant rats are administered drug-loaded NPs via tail vein. Administration is as described for EtOH and control rats.

[0132] The present inventors developed and optimized NPs by changing NP composition to enhance the targeting capability and drug loading and stability of the NPs. NPs characteristics including size, zeta potential, poly dispersity, loading capacity, and encapsulation efficiency are determined. The size and morphology of content-loaded NPs have been measured using 200Kv Hitachi H-8100 analytical transmission electron microscope (TEM). Size, size distribution, and zeta potential will be measured by using Malvern Zetasizer. Drugs are detected by an Agilent 1290 Infinity II UHPLC system. Drug encapsulation efficiency and loading capacity are calculated as previously described by the present inventors. (See, e.g., Zu Y, Overby H, Ren G, Fan Z, Zhao L, Wang S. / raas-resveratrol liposomes and lipid nanocarriers: Comparison of characteristics and inducing browning of white adipocytes. Colloids Surf B Biointerfaces. 164:414-423. 2018., Zu Y, Zhao L, Hao L, Mechref Y, Zabet-Moghaddam M, Keyel PA, Abbasi M, Wu D, Dawson JA, Zhang R, Nie S, Moustaid-Moussa N, Kolonin MG, Daquinag AC, Brandi L, Warraich I, San Francisco SK, Sun X, Fan Z, Wang S. Browning white adipose tissue using adipose stromal cell- targeted / ra / / .s-Resveratrol -loaded nanoparticles for combating obesity. J Control Release. 333:339-351. 2021, relevant portions of the materials and methods are incorporated herein by reference).

[0133] During the NP preparation, the compound / drug to be encapsulated and a-tocopherol acetate are packed into the hydrophobic core of NPs during sonication in hot water. The hydrophobic core of NPs can also be modified to enhance loading capacity and stability by replacing a-tocopherol acetate with another vitamin E form like a-tocopherol nicotinate (Sigma T5134, melting temperature about 38°C) or other lipids. Melting temperature goal of the hydrophobic core is close to body temperature. Such an optimized NP structure prevents the activators from leaking in the body before being taken up by placental trophoblasts. Any leaked compound, e.g., NFAT3 and Nrf2 activators will act like free activators, which can be quickly metabolized to become inactive.

[0134] Target specificity in timed pregnant rats: The inventors encapsulated 1,1 '-dioctadecyl - 3,3,3',3'-tetramethyl indotricarbocyanine iodide (DiR), a near infrared dye, into both targeted and non-targeted NPs. Both these DiR-labeled NPs are injected into pregnant rats via tail veins (FIG. 8). The biodistribution of DiR-labeled NPs is measured using an IVIS® Lumina XR imaging system at 1, 2, 4, 6, and 24 hours post-injection (FIG. 8).

[0135] FIG. 8 shows the placental / fetal nanoparticle localization in hours post material injection. Often, rats are too large for whole body scans, thus injected rats are sacrificed at time points, maternal organs and fetuses along with their placenta are scanned to determine the NP content biodistribution over these periods. These tissues are assayed for the encapsulated compound delivered. FIG. 8 confirms specific targeting to placenta with no fluorescence / nanoparticles in the fetus.

[0136] Human trophoblast models (HTR8 / SVneo and BeWo cells) in vitro models.

[0137] In vitro studies were used on differentiated human syncytiotrophoblast BeWo cells and HTR-8 / SVneo cell line as a cytotrophoblast model (potentially early gestation) to support the undifferentiated BeWo cell model and to connect to human relevance. BeWo is a cell line isolated from the placenta of a patient with choriocarcinoma and is generally considered an ideal model for studying trophoblast transcellular transport of amino acids, vitamins, folic acid, fatty acids, choline, serotonin etc. These cells differentiate into syncytiotrophoblasts (STB) by exposure to 8- Br-cAMP or forskolin.

[0138] FIG. 5A shows the differentiation of the BeWo cells after a 48-hour Forskolin treatment. The differentiation is monitored by expression of syncytialization-related genes like OVOL1 and CGB using comparative Ct method. HTR-8 / SVneo is a cell line derived from human placental explant cultures and immortalized by Simian virus 40 (SV40) large T antigens. Cells exhibit primary cytotrophoblast phenotypic marker response patterns similar to trophoblasts and are widely regarded as a model for normal cytotrophoblast biology.

[0139] Transient transfection and NFAT3 and Nrf2 luciferase reporter assays: The cells were transiently transfected using Xtreme GENE HP DNA transfection reagent (Roche Applied Science, IN) or TransIT-2020 (Minis Bio LLC, WI) with either control / blank vector (pREP / pEF for NF AT c4 and Nrf2) and exogenous overexpression of NFATc4 / Ar / 2 cDNA constructs (pREP- NFATc4 / pEF- / 2). For transcription activation using luciferase reporter activity assays the cells are co-transfected with pGL4.30 [luc2P / NFAT-RE / Hygro] reporter plasmid for NFATc4 or 4x ARE reporter plasmid for Nrf2 and renilla luc (pRL-TK). Gene reporter activity was determinedusing dual luciferase reporter assay system (Promega, Madison, WI). Luciferase activity was determined as the ratio of firefly luciferase to renilla luciferase corrected to protein.

[0140] Reactive oxygen species (ROS) detection was determined using DCF-DA using fluorimetry. DCF-DA is used for ROS detection with subsequent fluorimetry analysis.

[0141] GSH / GSSG Gio assay: Luminescence-based GSH / GSSG Gio assay experiments are carried out as previously described (see, e.g., Shanmugam S, Patel D, Rodriguez AL, Walchale A, Liu X, Bergeson SE, Mahimainathan L, Narasimhan M, Henderson GI. Ethanol inhibition of undifferentiated rat neural progenitor cell replication can be prevented by chlorogenic acid via the NFATc4 / CSE signaling pathway. Alcohol Clin Exp Res. 47, 1530-1543, 2023, relevant materials and methods incorporated herein by reference). Cells and tissues are processed for total glutathione (GSH) and oxidized glutathione (GSSG) in parallel reactions. N-Ethylmaleimide (NEM) treatment locks glutathione in its reduced state and intact GSSG that is left unblocked by NEM is reduced to GSH followed by the addition of luciferin generation reagent containing DTT and glutathione-S-transferase. Both GSH and GSSG levels are determined against standard GSH.

[0142] NFAT3, and Nrf2 activators are encapsulated into the NPs. The animal study will include the following groups saline, Void nanoparticle (Void NP, no drug), plCSA ligand conjugated NPs (plCSA-NP), free drug (the activators), drug encapsulated NPs, and drug encapsulated plCSA NPs (with trans-Resveratrol as shown in FIG. 10).

[0143] FIG. 10 shows that the nanoparticles (NP) successfully targeted reservatrol, which lead to an increase in targeted gene expression in placenta in vivo.

[0144] FIGS. 11A and 11B show that specific placental delivery of resveratrol can significantly normalize the ethanol-induced decrease of mRNA expression of the vital cystine transporter (Slc7all / xCT) in both rat placenta (FIG. 11 A) and fetal brain (FIG. 1 IB). There is an extensive literature connecting this transporter to enabling control of cellular redox homeostasis. In vivo ethanol (EtOH) exposure is from gestation days (GD) 11 to 20 (second trimester equivalent). The NPs containing Resveratrol (NP) were injected i.v. (tail vein) on GD18 and the placenta and corresponding fetal brain were harvested on GD20. Targeted Resveratrol-NP treatment significantly rescued expression of the Cystine transporter (Slc7al 7 / xCT), vital to control of redox homeostasis, in both placenta and fetal brain.

[0145] FIGS. 12A and 12B shows specific placental delivery of resveratrol prevents the Ethanol (EtOH)-induced oxidative damage that the inventors and others have shown elicits placenta dysfunction and fetal neuron apoptotic death. Most importantly, this is compelling evidence that the nanoparticle delivery system can rescue the fetal brain by specifically targeting the placenta.In vivo ethanol (EtOH) exposure is from gestation days (GD) 11 to 20 (second trimester equivalent). The NPs containing Resveratrol (NP) were injected i.v. (tail vein) on GDI 8 and the placenta and corresponding fetal brain were harvested on GD20. 4-hydroxynonenal (4-HNE) is a highly toxic lipid peroxidation end product which forms stable protein adducts. Ethanol-induced 4-HNE production has been shown to play a mechanistic role in fetal brain proapoptotic cell death responses to ethanol. Measuring 4-HNE adducts is a widely accepted measure of oxidative damage.

[0146] Fetal brain rescue by specifically normalizing placental function: Determinations will be as detailed in as follows. Measures of EtOH-Mediated Cell Damage: Placental and fetal cortical neuron damage will be assessed by measures of oxidative damage (HNE protein adducts, Malondialdehyde, DNA fragmentation, PARP-1 expression); apoptosis markers (AIF), cytochrome C release, activated caspase-3, and / or Nrf2 expression.

[0147] Loss-and-Gain of function experiments: siRNA or Morpholino mediated Knockdown of Nrf2 (140) and NFAT3 for studying loss of function in the in vivo models. Specific inhibition of KEAP1 (Nrf2 is bound to Keapl in the cytoplasm and targeted for ubiquitination are used, and subsequent proteasomal degradation) using siRNA helps in the rescue strategies.

[0148] Support for the BeWo STB Model. EtOH decreased the expression of the two transcription factors NFAT3 and Nrf2 and their targets Cth, SLC1A1 and SLC7A11 in differentiated BeWo cells (syncytiotrophoblast (STB) cell model-).

[0149] FIGS. 6A to 6C show that EtOH decreased expression of Cth, NFAT3, Nrf2 and the two transporters xCT (cystine) / SLC7Al 1 and EAAC1 (cysteine) / SLC1A1 in sy ncy ti otrophobl asts / S TB .

[0150] Trans-resveratrol delivered to STB cells in vitro and placenta in vivo by NPs. The inventors treated via the functioning nanoparticle systems (NP) loaded with a Nrf2 activator (trans- Resveratrol) and the targeting peptide (placental chondroitin sulfate A (plCSA)-targeting peptide). Trans-resveratrol content was evaluated in vitro STB cells treated for 4h and in placental tissue of pregnant rats in vivo after treatment with Void and Targeted NP for 24h using HPLC.

[0151] FIGS. 5B and 9 show the cellular Trans-resveratrol content in STB cells treated with targeted NP compared to non-targeted (void) was ~2.7-fold higher. Trans-resveratrol content in the rat placenta was measured 24h post tail vein inj ection. The Trans-resveratrol content was ~3.4- fold higher in NP targeted placenta when compared with non-targeted (Void NP).

[0152] FIGS. 7 A and 7B show that NPs loaded with trans-Resveratrol increased the target gene expression in differentiated BeWo cells. Trans-resveratrol activated the transcription factorNFE2L2, eliciting increases in the 3 intervention targets, the Cys / CySS transporters (SLC1A1 / EAAC1 and SLC7Al l / xCT) and Clh. the rate limiting enzyme in the transsulfuration pathway.

[0153] FIG. 4 shows a comparison of expression of nanoparticles using LI (SEQ ID NO: 2), and L2 (SEQ ID NO: 1) as the targeting peptide, using expression of Nrf2 in Rat Placenta.

[0154] FIG. 3 shows a comparison of expression of nanoparticles using LI (SEQ ID NO: 2), and L2 (SEQ ID NO: 1) as the targeting peptide, using expression of Nrf2 in HTR8 cells.

[0155] Embodiments

[0156] Embodiment 1. A peptide that targets placental chondroitin sulfate A (placental CSA), wherein an amino acid sequence of the peptide is as set forth in SEQ ID NO: 1 (EDVKDINFDTKEKFLAGLIVSFHEGKC).

[0157] Embodiment 2. A targeted delivery system that targets placenta-like chondroitin sulfate A, wherein the targeted delivery system comprises the peptide of embodiment 1.

[0158] Embodiment 3. The targeted delivery system of embodiment 2, wherein the peptide is not EDVKDINFDTKEKFLAGCLIVSFHEGK (SEQ ID NO: 2).

[0159] Embodiment 4. The targeted delivery system of embodiment 2 or 3, wherein the peptide is conjugated to a phospholipid and the conjugated peptide-phospholipid is incorporated into a particle.

[0160] Embodiment 5. The targeted delivery system of embodiment 4, wherein the particle is a micro- or nano-particle further comprises one or more of the following: a hydrophobic core, a surfactant, a phospholipid, a hydrophobic polymer, an amphiphilic macromolecule grafted to the peptide, or an amphiphilic macromolecule interspersed in a lipid monolayer or bilayer.

[0161] Embodiment 6. The targeted delivery system of embodiment 4, wherein the particle is spherical and has a diameter of nanometers or micrometers.

[0162] Embodiment 7. The targeted delivery system of embodiment 4, wherein the particle further comprises one or more active agents or biomarkers for placental targeting.

[0163] Embodiment 8. The targeted delivery system of any one of embodiments 2 to 7, wherein the one or more active agents are selected for treating a placental disease or a disease that traverse a placental barrier, or not traverse the placental barrier, selected from active agents to treat Fetal Alcohol Spectrum Disorder (FASD), second-trimester ethanol exposure, preeclampsia, gestational diabetes, intrauterine growth restriction, target vaccines or prophylactic agents to enhance fetal immunity, target placental tumors or abnormalities, placenta associated syndromes,placental abruption, placenta previa, small for gestational age, preterm or still birth; normalize redox homeostasis, glutathione synthesis, or activate a transsulfuration pathway selected from chlorogenic acid and caffeic acid, and derivatives thereof; NFATc4 / NFAT3 transcriptional regulators; regulators of cysteine transport (EAAC1 (SLC1A1)) and cystine transport (Xc- (SLC7A11)); activation of Nrf2 (nuclear factor E2-related factor 2) selected from resveratrol and KI696; compounds supplying cysteine for glutathione synthesis selected from N-acetyl cysteine and derivatives thereof; proangiogenic factors selected from VEGF (VEGF-A mRNA), Netrin-1 and Notchl signaling, activators of glutathione peroxidase; resveratrol; or selenium.

[0164] Embodiment 9. The targeted delivery system of any one of embodiments 2 to 7, wherein the one or more biomarkers is selected from: a contrast agent, a fluorescence tracer, a photothermal conversion reagent, a molecule to study placental physiology or drug transport during pregnancy, or combinations thereof.

[0165] Embodiment 10. The targeted delivery system of any one of embodiments 2 to 5, wherein the amphiphilic macromolecule is a polyethylene glycol derivatized phospholipid, the polyethylene glycol derivatized phospholipid being passed from polyethylene glycol and its derivatives.

[0166] Embodiment 11. The targeted delivery system of any one of embodiments 2 to 10, wherein the amphiphilic macromolecule is a polyethylene glycol-derivatized phospholipid, and the polyethylene glycol-derivatized phospholipid is passed through a polyethylene glycol and a derivative thereof, and one or more viscous or viscosity enhancing molecules selected from at least one of polyvinyl alcohol, glucose, hyaluronic acid, or gelatin.

[0167] Embodiment 12. The targeted delivery system of any one of embodiments 2 to 11, wherein the targeted delivery system further comprises at least one of: one or more proteins, one or more carbohydrates, one or more lipids, or one or more small molecules.

[0168] Embodiment 13. The targeted delivery system of any one of embodiments 2 to 12, wherein the targeted delivery system further comprises one or more excipients or carriers selected from: pharmaceutical carriers, inorganic nanomaterials, diluents, adhesives, buffers, or salts.

[0169] Embodiment 14. The targeted delivery system of any one of embodiments 7 to 13, wherein the targeted delivery system delivers a second active agent or a pregnancy drug for treating gestational diabetes, treating pregnancy syndrome, treating intrauterine growth retardation, anti-epilepsy, anti-inflammatory, premature delivery, prevents premature rupture of membranes, a polypeptide drug, and or a gene therapy.

[0170] Embodiment 15. A method of preparing a targeted delivery system, comprising the steps of:

[0171] conjugating a peptide EDVKDINFDTKEKFLAGLIVSFHEGKC of SEQ ID NO: 1 to a phospholipid and the conjugated peptide-phospholipid is incorporated into a particle;

[0172] mixing the conjugated peptide with one or more phospholipids to form a monolayer or bilayer;

[0173] adding one or more surfactants, carriers, active agents, or payloads into a mixture; and

[0174] ultrasonically treating the mixture to form active agent loaded particles.

[0175] Embodiment 16. The method of embodiment 15, wherein the peptide is not EDVKDINFDTKEKFLAGCLIVSFHEGK (SEQ ID NO: 2).

[0176] Embodiment 17. The method of embodiment 15 or 16, wherein the peptide is attached or conjugated to the one or more phospholipid and the conjugated peptide-phospholipid is incorporated into a particle.

[0177] Embodiment 18. The method of any one of embodiments 15 to 17, wherein the particle further comprises one or more of the following: a hydrophobic core, a surfactant, a phospholipid, a hydrophobic polymer, an amphiphilic macromolecule grafted to the peptide, or an amphiphilic macromolecule interspersed in a lipid monolayer or bilayer.

[0178] Embodiment 19. The method of any one of embodiments 15 to 18, wherein the particle is spherical and has a diameter of nanometers or micrometers.

[0179] Embodiment 20. The method of any one of embodiments 15 to 19, wherein the particle further comprises one or more active agents or biomarkers for placental targeting.

[0180] Embodiment 21. The method of embodiment 20, wherein the one or more active agents are selected for treating a placental disease or a disease that traverse a placental barrier, or not traverse the placental barrier, selected from active agents to treat Fetal Alcohol Spectrum Disorder (FASD), second-trimester ethanol exposure, preeclampsia, gestational diabetes, intrauterine growth restriction, target vaccines or prophylactic agents to enhance fetal immunity, target placental tumors or abnormalities, placenta associated syndromes, placental abruption, placenta previa, small for gestational age, preterm or still birth; normalize redox homeostasis, glutathione synthesis, or activate a transsulfuration pathway selected from chlorogenic acid and caffeic acid, and derivatives thereof; NFATc4 / NFAT3 transcriptional regulators; regulators of cysteine transport (EAAC1 (SLC1 Al)) and cystine transport (Xc- (SLC7A11)); activation of Nrf2 (nuclear factor E2-related factor 2) selected from resveratrol and KI696; compounds supplyingcysteine for glutathione synthesis selected from N-acetyl cysteine and derivatives thereof; proangiogenic factors selected from VEGF (VEGF-A mRNA), Netrin-1 and Notch 1 signaling, activators of glutathione peroxidase; resveratrol; or selenium.

[0181] Embodiment 22. The method of embodiment 20, wherein the one or more biomarkers is selected from at least one of: a contrast agent, a fluorescence tracer, a photothermal conversion reagent, a molecule to study placental physiology or drug transport during pregnancy, or combinations thereof.

[0182] Embodiment 23. The method of embodiment 18, wherein the amphiphilic macromolecule is a polyethylene glycol derivatized phospholipid, the polyethylene glycol derivatized phospholipid being passed from polyethylene glycol and its derivatives.

[0183] Embodiment 24. The method of embodiment 18, wherein the amphiphilic macromolecule is a polyethylene glycol-derivatized phospholipid, and the polyethylene glycol- derivatized phospholipid is passed through a polyethylene glycol and a derivative thereof, and one or more viscous or viscosity enhancing molecules selected from at least one of polyvinyl alcohol, glucose, hyaluronic acid, or gelatin.

[0184] Embodiment 25. The method of any one of embodiments 15 to 24, wherein the targeted delivery system further comprises at least one of: one or more proteins, one or more carbohydrates, one or more lipids, or one or more small molecules.

[0185] Embodiment 26. The method of any one of embodiments 15 to 25, wherein the targeted delivery system further comprises one or more excipients or carriers selected from: pharmaceutical carriers, inorganic nanomaterials, diluents, adhesives, buffers, or salts.

[0186] Embodiment 27. The method of any one of embodiments 15 to 26, wherein the targeted delivery system delivers a pregnancy drug for treating gestational diabetes, treating pregnancy syndrome, treating intrauterine growth retardation, anti-epilepsy, anti-inflammatory, premature delivery, prevents premature rupture of membranes, a polypeptide drug, and or a gene therapy.

[0187] Embodiment 28. A method of treating a pregnancy disease, a fetal substance abuse disorder, or targeting the placenta comprising:

[0188] identifying a patient in need of treatment for the pregnancy disease or in need of targeting the placenta; and

[0189] providing the patient with a therapeutically effective amount of a pharmaceutical composition comprising the peptide of embodiment 1.

[0190] Embodiment 29. The method of embodiment 28, wherein the pregnancy disease or targeting a placenta is selected from Fetal Alcohol Spectrum Disorder (FASD), preeclampsia, gestational diabetes, intrauterine growth restriction, target vaccines or prophylactic agents to enhance fetal immunity, target placental tumors or abnormalities, placenta associated syndromes, placental abruption, placenta previa, small for gestational age, preterm or still birth, or combinations thereof.

[0191] Embodiment 30. The method of embodiment 28 or 29, further comprising one or more active agents are selected for treating a placental disease or a disease that traverse a placental barrier, or not traverse the placental barrier, selected from active agents to treat Fetal Alcohol Spectrum Disorder (FASD), second-trimester ethanol exposure, preeclampsia, gestational diabetes, intrauterine growth restriction, target vaccines or prophylactic agents to enhance fetal immunity, target placental tumors or abnormalities, placenta associated syndromes, placental abruption, placenta previa, small for gestational age, preterm or still birth; normalize redox homeostasis, glutathione synthesis, or activate a transsulfuration pathway selected from chlorogenic acid and caffeic acid, and derivatives thereof; NFATc4 / NFAT3 transcriptional regulators; regulators of cysteine transport (EAAC1 (SLC1A1)) and cystine transport (Xc- (SLC7A11)); activation of Nrf2 (nuclear factor E2-related factor 2) selected from resveratrol and KI696; compounds supplying cysteine for glutathione synthesis selected from N-acetyl cysteine and derivatives thereof; proangiogenic factors selected from VEGF (VEGF-A mRNA), Netrin-1 and Notchl signaling, activators of glutathione peroxidase; resveratrol; or selenium.

[0192] Embodiment 31. A pharmaceutical preparation for treating a pregnancy disease or targeting a placenta, wherein the pharmaceutical preparation for treating a pregnancy disease or targeting the placenta comprises the peptide of embodiment 1 or the targeted delivery system of embodiment 2.

[0193] Embodiment 32. The pharmaceutical preparation of embodiment 31, wherein the peptide is used to target active agents to treat Fetal Alcohol Spectrum Disorder (FASD), second- trimester ethanol exposure, preeclampsia, gestational diabetes, intrauterine growth restriction, target vaccines or prophylactic agents to enhance fetal immunity, target placental tumors or abnormalities, placenta associated syndromes, placental abruption, placenta previa, small for gestational age, preterm or still birth, or combinations thereof.

[0194] Embodiment 33. The pharmaceutical preparation of embodiment 31 or 32, further comprising one or more active agents are selected for treating a placental disease or a disease that traverse a placental barrier, or not traverse the placental barrier, selected from active agents to treat Fetal Alcohol Spectrum Disorder (FASD), preeclampsia, gestational diabetes, intrauterinegrowth restriction, target vaccines or prophylactic agents to enhance fetal immunity, target placental tumors or abnormalities, placenta associated syndromes, placental abruption, placenta previa, small for gestational age, preterm or still birth; normalize redox homeostasis, glutathione synthesis, or activate a transsulfuration pathway selected from chlorogenic acid and caffeic acid, and derivatives thereof; NFATc4 / NFAT3 transcriptional regulators; regulators of cysteine transport (EAAC1 (SLC1 Al)) and cystine transport (Xc- (SLC7A11)); activation of Nrf2 (nuclear factor E2 -related factor 2) selected from resveratrol and KI696; compounds supplying cysteine for glutathione synthesis selected from N-acetyl cysteine and derivatives thereof; proangiogenic factors selected from VEGF (VEGF-A mRNA), Netrin-1 and Notch 1 signaling, activators of glutathione peroxidase; resveratrol; or selenium.

[0195] It is contemplated that any aspects of the disclosure discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the disclosure, and vice versa. Furthermore, compositions of the disclosure can be used to achieve methods of the disclosure.

[0196] It will be understood that particular aspects described herein are shown by way of illustration and not as limitations of the disclosure. The principal features of this disclosure can be employed in various aspects without departing from the scope of the disclosure. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this disclosure and are covered by the claims.

[0197] All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0198] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

[0199] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open- ended and do not exclude additional, unrecited elements or method steps. In aspects of any of the compositions and methods provided herein, “comprising” may be replaced with “consisting essentially of’ or “consisting of’. As used herein, the phrase “consisting essentially of’ requires the specified integer(s) or steps as well as those that do not materially affect the character or function of the claimed invention. As used herein, the term “consisting” is used to indicate the presence of the recited integer (e.g., a feature, an element, a characteristic, a property, a method / process step or a limitation) or group of integers (e.g., feature(s), element(s), characteristic(s), propertie(s), method / process steps or limitation(s)) only.

[0200] The term “or combinations thereof’ as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof’ is intended to include at least one of A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0201] As used herein, words of approximation such as, without limitation, “about”, "substantial" or "substantially" refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present. The extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skilled in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature. In general, but subject to the preceding discussion, a numerical value herein that is modified by a word of approximation such as “about” may vary from the stated value by at least ±1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.

[0202] Additionally, the section headings herein are provided for consistency with the suggestions under 37 CFR 1.77 or otherwise to provide organizational cues. These headings shall not limit or characterize the disclosure(s) set out in any claims that may issue from this disclosure. Specifically, and by way of example, although the headings refer to a “Field of Invention,” such claims should not be limited by the language under this heading to describe the so-called technicalfield. Further, a description of technology in the “Background of the Invention” section is not to be construed as an admission that technology is prior art to any disclosure(s) in this disclosure. Neither is the “Summary” to be considered a characterization of the disclosure(s) set forth in issued claims. Furthermore, any reference in this disclosure to “invention” in the singular should not be used to argue that there is only a single point of novelty in this disclosure. Multiple inventions may be set forth according to the limitations of the multiple claims issuing from this disclosure, and such claims accordingly define the invention(s), and their equivalents, that are protected thereby. In all instances, the scope of such claims shall be considered on their own merits in light of this disclosure but should not be constrained by the headings set forth herein.

[0203] All of the compositions and / or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred aspects, it will be apparent to those of skill in the art that variations may be applied to the compositions and / or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

[0204] To aid the Patent Office, and any readers of any patent issued on this application in interpreting the claims appended hereto, applicants wish to note that they do not intend any of the appended claims to invoke paragraph 6 of 35 U.S.C. § 112, U.S.C. § 112 paragraph (f), or equivalent, as it exists on the date of filing hereof unless the words “means for” or “step for” are explicitly used in the particular claim.

[0205] For each of the claims, each dependent claim can depend both from the independent claim and from each of the prior dependent claims for each and every claim so long as the prior claim provides a proper antecedent basis for a claim term or element.REFERENCES

[0206] 1. Henderson GI, Devi BG, Perez A, Schenker S. In utero ethanol exposure elicits oxidative stress in the rat fetus. Alcohol Clin Exp Res 19:714-720, 1995. PMID:7573798

[0207] 2. Hamby-Mason RL, Mason PA, Schenker S and Henderson GI. Histochemical Localization of Hydrogen Peroxide and Superoxide Anion Radical in Neonatal Rat Brain. Method Find Exp Clin Pharmacol 20:743-748, 1998. PMID: 10022027

[0208] 3. Ramachandran V, Perez A, Chen J, Senthil D, Schenker S and Henderson GI. In Utero Ethanol Exposure Causes Mitochondrial Dysfunction Which Can Result in Apoptotic Cell Death in Fetal Brain: A Potential Role for 4-Hydroxynonenal. Alcohol Clin Exp Res 25:862-871, 2001. PMID: 1141072327.

[0209] 4. Ramachandran V, Watts LT, Maffi S, Chen JJ, Schenker S, Henderson GI. Ethanol- Induced Oxidative Stress Precedes Mitochondrially-Mediated Apoptotic Death of Cultured Fetal Cortical Neurons. J Neuroscience Research V74: 577-588, 2003. PMID: 14598302

[0210] 5. Maffi S, Rathimnam M, Cherian, Pate W. Hamby-Mason R, Schenker S. Henderson GI. Glutathione Content Mediates the Vulnerability of Cultured Fetal Cortical Neurons to Ethanol- Induced Apoptosis. J Neurosci Res 86: 1064-1076, 2008. PMID: 18058941

[0211] 6. Narasimhan M, Mahimainathan L, Rathinam ML, Riar AK, Henderson GI. Overexpression of Nrf2 protects cerebral cortical neurons from ethanol -induced apoptotic death. Molecular Pharmacology, 80: 988-999, 2011. PMID: 21873460

[0212] 7. Patel D., Rathinam M., Jarvis C., Mahimainathan L., Henderson G., Narasimhan M., G. Henderson. Role for Cystathionine Lyase (CSE) in an Ethanol (E)-Induced Lesion in Fetal Brain GSH Homeostasis. Int. J. Mol. Sci. 19, 1537, 2018.

[0213] 8. Shanmugam S, Patel D, Rodriguez AL, Walchale A, Liu X, Bergeson SE, Mahimainathan L, Narasimhan M, Henderson GI. Ethanol inhibition of undifferentiated rat neural progenitor cell replication can be prevented by chlorogenic acid via the NFATc4 / CSE signaling pathway. Alcohol: Clinical and Experimental Research, 47, 1530-1543, 2023.

[0214] 9. Shanmugam S, Patel D, Wolpert JM, Keshvani C, Liu X, Bergeson SE, Kidambi S, Mahimainathan L, Henderson GI, Narasimhan M. Ethanol Impairs NRF2 / Anti oxidant and Growth Signaling in the Intact Placenta In Vivo and in Human Trophoblasts. Biomolecules. Oct 30;9(l 1), 2019.

[0215] 10. Patel D, Mahimainathan L, Narasimhan M, Rathinam M, Henderson G. Ethanol (E) Impairs Fetal Brain GSH Homeostasis by Inhibiting Excitatory Amino-Acid Carrier 1 (EAAC1)- Mediated Cysteine Transport. Int J Mol Sci. 18(12):2596. 2017.

[0216] 11. Narasimhan M, Rathinam M, Riar A, Patel D, Mummidi S, Yang HS, Colburn NH, Henderson GI, Mahimainathan L. Programmed cell death 4 (PDCD4): a novel player in ethanol- mediated suppression of protein translation in primary cortical neurons and developing cerebral cortex. Alcohol Clin Exp Res. 2013 Jan;37(l):96-109.

[0217] 12. Zu Y, Overby H, Ren G, Fan Z, Zhao L, Wang S. Resveratrol liposomes and lipid nanocarriers: Comparison of characteristics and inducing browning of white adipocytes. Colloids Surf B Biointerfaces. 164:414-423. 2018.

[0218] 13. Zhang J, Nie S, Zu Y, Abbasi M, Cao J, Li C, Wu D, Labib S, Brackee G, Shen CL, Wang S. Antiatherogenic effects of CD36-targeted epigallocatechin gallate-loaded nanoparticles.J Control Release. 10; 303:263-273. 2019.

[0219] 14. Zu Y, Zhao L, Hao L, Mechref Y, Zabet-Moghaddam M, Keyel PA, Abbasi M, Wu D, Dawson JA, Zhang R, Nie S, Moustaid-Moussa N, Kolonin MG, Daquinag AC, Brandi L, Warraich I, San Francisco SK, Sun X, Fan Z, Wang S. Browning white adipose tissue using adipose stromal cell targeted resveratrol -loaded nanoparticles for combating obesity. J Control Release. 333:339-351.2021.

Claims

What is claimed is:

1. A peptide that targets placental chondroitin sulfate A (placental CSA), wherein an amino acid sequence of the peptide is as set forth in SEQ ID NO: 1 (EDVKDINFDTKEKFLAGLIVSFHEGKC).

2. A targeted delivery system that targets placenta-like chondroitin sulfate A, wherein the targeted delivery system comprises the peptide of claim 1.

3. The targeted delivery system of claim 2, wherein the peptide is not EDVKDINFDTKEKFLAGCLIVSFHEGK (SEQ ID NO: 2).

4. The targeted delivery system of claim 2, wherein the peptide is conjugated to a phospholipid and the conjugated peptide-phospholipid is incorporated into a particle.

5. The targeted delivery system of claim 4, wherein the particle is a micro- or nano-particle further comprising one or more of the following: a hydrophobic core, a surfactant, a phospholipid, a hydrophobic polymer, an amphiphilic macromolecule grafted to the peptide, or an amphiphilic macromolecule interspersed in a lipid monolayer or bilayer.

6. The targeted delivery system of claim 4, wherein the particle is spherical and has a diameter of nanometers or micrometers.

7. The targeted delivery system of claim 4, wherein the particle further comprises one or more active agents or biomarkers for placental targeting.

8. The targeted delivery system of claim 7, wherein the one or more active agents are selected for treating a placental disease or a disease that traverse a placental barrier, or not traverse the placental barrier, selected from active agents to treat Fetal Alcohol Spectrum Disorder (FASD), second-trimester ethanol exposure, preeclampsia, gestational diabetes, intrauterine growth restriction, target vaccines or prophylactic agents to enhance fetal immunity, target placental tumors or abnormalities, placenta associated syndromes, placental abruption, placenta previa, small for gestational age, preterm or still birth; normalize redox homeostasis, glutathione synthesis, or activate a transsulfuration pathway selected from chlorogenic acid and caffeic acid, and derivatives thereof; NFATc4 / NFAT3 transcriptional regulators; regulators of cysteine transport (EAAC1 (SLC1 Al)) and cystine transport (Xc- (SLC7A11)); activation of Nrf2 (nuclear factor E2-related factor 2) selected from resveratrol and KI696; compounds supplying cysteine for glutathione synthesis selected from N-acetyl cysteine and derivatives thereof; proangiogenic factors selected from VEGF (VEGF-A mRNA), Netrin-1 and Notch 1 signaling, activators of glutathione peroxidase; resveratrol; or selenium.

9. The targeted delivery system of claim 7, wherein the one or more biomarkers is selected from: a contrast agent, a fluorescence tracer, a photothermal conversion reagent, a molecule to study placental physiology or drug transport during pregnancy, or combinations thereof.

10. The targeted delivery system of claim 5, wherein the amphiphilic macromolecule is a polyethylene glycol derivatized phospholipid, the polyethylene glycol derivatized phospholipid being passed from polyethylene glycol and its derivatives.

11. The targeted delivery system of claim 10, wherein the amphiphilic macromolecule is a polyethylene glycol -derivatized phospholipid, and the polyethylene glycol -derivatized phospholipid is passed through a polyethylene glycol and a derivative thereof, and one or more viscous or viscosity enhancing molecules selected from at least one of polyvinyl alcohol, glucose, hyaluronic acid, or gelatin.

12. The targeted delivery system of claim 2, wherein the targeted delivery system further comprises at least one of: one or more proteins, one or more carbohydrates, one or more lipids, or one or more small molecules.

13. The targeted delivery system of claim 2, wherein the targeted delivery system further comprises one or more excipients or carriers selected from: pharmaceutical carriers, inorganic nanomaterials, diluents, adhesives, buffers, or salts.

14. The targeted delivery system of claim 7, wherein the targeted delivery system delivers a second active agent or a pregnancy drug for treating gestational diabetes, treating pregnancy syndrome, treating intrauterine growth retardation, anti-epilepsy, anti-inflammatory, premature delivery, prevents premature rupture of membranes, a polypeptide drug, and or a gene therapy.

15. A method of preparing a targeted delivery system, comprising the steps of: conjugating a peptide EDVKDINFDTKEKFLAGLIVSFHEGKC of SEQ ID NO: 1 to a phospholipid and the conjugated peptide-phospholipid is incorporated into a particle; mixing the conjugated peptide with one or more phospholipids to form a monolayer or bilayer; adding one or more surfactants, carriers, active agents, or payloads into a mixture; and ultrasonically treating the mixture to form active agent loaded particles.

16. The method of claim 15, wherein the peptide is not EDVKDINFDTKEKFLAGCLIVSFHEGK (SEQ ID NO: 2).

17. The method of claim 15, wherein the peptide is attached or conjugated to the one or more phospholipid and the conjugated peptide-phospholipid is incorporated into a particle.

18. The method of claim 17, wherein the particle further comprises one or more of the following: a hydrophobic core, a surfactant, a phospholipid, a hydrophobic polymer, anamphiphilic macromolecule grafted to the peptide, or an amphiphilic macromolecule interspersed in a lipid monolayer or bilayer.

19. The method of claim 15, wherein the particle is spherical and has a diameter of nanometers or micrometers.

20. The method of claim 15, wherein the particle further comprises one or more active agents or biomarkers for placental targeting.

21. The method of claim 20, wherein the one or more active agents are selected for treating a placental disease or a disease that traverse a placental barrier, or not traverse the placental barrier, selected from active agents to treat Fetal Alcohol Spectrum Disorder (FASD), second- trimester ethanol exposure, preeclampsia, gestational diabetes, intrauterine growth restriction, target vaccines or prophylactic agents to enhance fetal immunity, target placental tumors or abnormalities, placenta associated syndromes, placental abruption, placenta previa, small for gestational age, preterm or still birth; normalize redox homeostasis, glutathione synthesis, or activate a transsulfuration pathway selected from chlorogenic acid and caffeic acid, and derivatives thereof; NFATc4 / NFAT3 transcriptional regulators; regulators of cysteine transport (EAAC1 (SLC1 Al)) and cystine transport (Xc- (SLC7A11)); activation of Nrf2 (nuclear factor E2 -related factor 2) selected from resveratrol and KI696; compounds supplying cysteine for glutathione synthesis selected from N-acetyl cysteine and derivatives thereof; proangiogenic factors selected from VEGF (VEGF-A mRNA), Netrin-1 and Notchl signaling, activators of glutathione peroxidase; resveratrol; or selenium.

22. The method of claim 20, wherein the one or more biomarkers is selected from at least one of: a contrast agent, a fluorescence tracer, a photothermal conversion reagent, a molecule to study placental physiology or drug transport during pregnancy, or combinations thereof.

23. The method of claim 18, wherein the amphiphilic macromolecule is a polyethylene glycol derivatized phospholipid, the polyethylene glycol derivatized phospholipid being passed from polyethylene glycol and its derivatives.

24. The method of claim 18, wherein the amphiphilic macromolecule is a polyethylene glycol-derivatized phospholipid, and the polyethylene glycol-derivatized phospholipid is passed through a polyethylene glycol and a derivative thereof, and one or more viscous or viscosity enhancing molecules selected from at least one of polyvinyl alcohol, glucose, hyaluronic acid, or gelatin.

25. The method of claim 15, wherein the targeted delivery system further comprises at least one of: one or more proteins, one or more carbohydrates, one or more lipids, or one or more small molecules.

26. The method claim 15, wherein the targeted delivery system further comprises one or more excipients or carriers selected from: pharmaceutical carriers, inorganic nanomaterials, diluents, adhesives, buffers, or salts.

27. The method of claim 15, wherein the targeted delivery system delivers a pregnancy drug for treating gestational diabetes, treating pregnancy syndrome, treating intrauterine growth retardation, anti -epilepsy, anti-inflammatory, premature delivery, prevents premature rupture of membranes, a polypeptide drug, and or a gene therapy.

28. A method of treating a pregnancy disease, a fetal substance abuse disorder, or targeting the placenta comprising: identifying a patient in need of treatment for the pregnancy disease or in need of targeting the placenta; and providing the patient with a therapeutically effective amount of a pharmaceutical composition comprising the peptide of claim 1.

29. The method of claim 28, wherein the pregnancy disease or targeting a placenta is selected from Fetal Alcohol Spectrum Disorder (FASD), preeclampsia, gestational diabetes, intrauterine growth restriction, target vaccines or prophylactic agents to enhance fetal immunity, target placental tumors or abnormalities, placenta associated syndromes, placental abruption, placenta previa, small for gestational age, preterm or still birth, or combinations thereof.

30. The method of claim 28, further comprising one or more active agents are selected for treating a placental disease or a disease that traverse a placental barrier, or not traverse the placental barrier, selected from active agents to treat Fetal Alcohol Spectrum Disorder (FASD), second-trimester ethanol exposure, preeclampsia, gestational diabetes, intrauterine growth restriction, target vaccines or prophylactic agents to enhance fetal immunity, target placental tumors or abnormalities, placenta associated syndromes, placental abruption, placenta previa, small for gestational age, preterm or still birth; normalize redox homeostasis, glutathione synthesis, or activate a transsulfuration pathway selected from chlorogenic acid and caffeic acid, and derivatives thereof; NFATc4 / NFAT3 transcriptional regulators; regulators of cysteine transport (EAAC1 (SLC1 Al)) and cystine transport (Xc- (SLC7A11)); activation of Nrf2 (nuclear factor E2 -related factor 2) selected from resveratrol and KI696; compounds supplying cysteine for glutathione synthesis selected from N-acetyl cysteine and derivatives thereof; proangiogenic factors selected from VEGF (VEGF-A mRNA), Netrin-1 and Notch 1 signaling, activators of glutathione peroxidase; resveratrol; or selenium.

31. A pharmaceutical preparation for treating a pregnancy disease or targeting a placenta, wherein the pharmaceutical preparation for treating a pregnancy disease or targeting the placenta comprises the peptide of claim 1 or the targeted delivery system of claim 2.

32. The pharmaceutical preparation of claim 31, wherein the peptide is used to target active agents to treat Fetal Alcohol Spectrum Disorder (FASD), second-trimester ethanol exposure, preeclampsia, gestational diabetes, intrauterine growth restriction, target vaccines or prophylactic agents to enhance fetal immunity, target placental tumors or abnormalities, placenta associated syndromes, placental abruption, placenta previa, small for gestational age, preterm or still birth, or combinations thereof.

33. The pharmaceutical preparation of claim 31, further comprising one or more active agents are selected for treating a placental disease or a disease that traverse a placental barrier, or not traverse the placental barrier, selected from active agents to treat Fetal Alcohol Spectrum Disorder (FASD), preeclampsia, gestational diabetes, intrauterine growth restriction, target vaccines or prophylactic agents to enhance fetal immunity, target placental tumors or abnormalities, placenta associated syndromes, placental abruption, placenta previa, small for gestational age, preterm or still birth; normalize redox homeostasis, glutathione synthesis, or activate a transsulfuration pathway selected from chlorogenic acid and caffeic acid, and derivatives thereof; NFATc4 / NFAT3 transcriptional regulators; regulators of cysteine transport (EAAC1 (SLC1 Al)) and cystine transport (Xc- (SLC7A11)); activation of Nrf2 (nuclear factor E2 -related factor 2) selected from resveratrol and KI696; compounds supplying cysteine for glutathione synthesis selected from N-acetyl cysteine and derivatives thereof; proangiogenic factors selected from VEGF (VEGF-A mRNA), Netrin-1 and Notchl signaling, activators of glutathione peroxidase; resveratrol; or selenium.

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