Nanoparticles and methods for targeting and treating vascular malformations

Abstract

Described herein are methods of selectively targeting a cell expressing upregulated PI3K activity in a subject, the method comprising: administering to the subject a plurality of nanoparticles having an average diameter between 60 nm to 200 nm, the nanoparticles comprising: a polymer; and, one or more a pharmaceutical agents. In embodiments, the methods are for the treatment of vascular malformations or cancer.

Description

Attorney Docket No. 035052 / 640791NANOPARTICLES AND METHODS FOR TARGETING AND TREATING VASCULAR MALFORMATIONSSTATEMENT OF GOVERNMENT SUPPORT

[0001] This invention was made with government support under Grant Number GM142944 awarded by the National Institutes of Health. The government has certain rights in the invention.FIELD

[0002] The subject matter described herein relates generally to nanoparticles and therapeutic uses thereof.BACKGROUND

[0003] Vascular malformations (VMs) are a class of rare genetic disorders associated with localized developmental abnormalities of venous, arterial, capillary, or lymphatic vessels. Vascular malformations are congenital anomalies of the blood and lymphatic vascular systems. The malformations are typically classified by the affected vessel type: capillary (CM), lymphatic (LM), venous (VM), and arteriovenous (AVM) malformations.

[0004] Histologically, VMs are characterized by complexes of structural lesions with enlarged, irregular lumens that are lined with vascular endothelial cells and surrounded by disorganized extracellular matrix. These lesions are often congenital, progress in severity over time, are associated with vascular obstruction and impaired drainage, and can be life threatening. Malformations harbor significant morbidity, including coagulopathy, thrombosis, pain, and location-specific functional limitations that worsen through puberty. Lesions of the central nervous system are associated with additional morbidity, including seizure, hemorrhage, and focal neurological deficits, and some slow-flow malformations are life-threatening due to extension into vital tissues such as the airway and cardiac tissue or due to severe coagulopathy.

[0005] Vascular malformations are often apparent at birth. VMs are associated with hematologic anomalies and painful intravascular coagulopathy. In combined lymphatic and venous malformations (LVMs), deep vein thrombosis can result in life-threatening pulmonary emboli. AVMs are associated with potentially life-threatening bleeding and heart failure. Another co-morbidity that affects all types of vascular malformation is hemorrhaging that can require1LEGAL02 / 47570581V1Attorney Docket No. 035052 / 640791 repeated transfusions. In sensitive locations such as the brain, VMs and AVMs can have neurologically devastating consequences that can lead to death.

[0006] LMs are congenital vascular anomalies that arise due to developmental dysplasia of the lymphatic network in utero. Although histologically benign, LMs can be progressive and may be debilitating due to obstruction or mass effect on vital functions or organs, risk for recurrent infection, and the negative psychosocial effects of disfigurement. Lymphatic dysfunction in LMs leads to defects in immune surveillance and lipid absorption leading to repeat infections and sepsis to malabsorption.

[0007] Patients often have significant morbidities and mortality and limited options. Treatment for VMs, especially for complex malformations that occur in multiple tissues is difficult and rarely curative. The current standard of care relies on surgery, sclerotherapy, and a limited repertoire of drugs. Thus, managing vascular malformations, such as, resection, embolization, sclerotherapy, and laser therapy are performed but can essentially only alleviate symptoms. Recurrence is common. There is no known cure. There are currently no FDA-approved pharmacotherapies although some off-label uses of approved drugs are being explored for VMs.

[0008] Nano- and therapeutic delivery technologies possess the potential to present solutions for addressing challenges like inadequate therapeutic accumulation in vascular malformations and reducing side effects such as systemic toxicity. However, while various techniques for synthesizing nanoparticles exist, many suffer from limitations such as inadequate scalability, low drug-loading efficiency, poor homogeneity of production, and imprecise size control.

[0009] There remains a need for improved nanoparticles and methods for targeting and treating vascular malformations. The subject matter described herein addresses these unmet needs and more.BRIEF SUMMARY

[0010] In certain embodiments, the subject matter described herein is directed to a method of selectively targeting a cell expressing upregulated PI3K activity in a subject in vivo, the method comprising: administering to the subject a plurality of nanoparticles having an average diameter from about 50 nm to about 220 nm, the nanoparticles comprising: one or more polymer layers; and,2LEGAL02 / 47570581V1Attorney Docket No. 035052 / 640791 one or more pharmaceutical agents.

[0011] In certain embodiments, the subject matter described herein is directed to a method of treating a disease associated with upregulated PI3K activity in a subject, the method comprising: administering to the subject a therapeutically effective amount of a pharmaceutical agent in a nanoparticle formulation, wherein the nanoparticle formulation comprises a plurality of nanoparticles having an average diameter of from about 50 nm to about 220 nm, and wherein the nanoparticles comprise a polymer and one or more pharmaceutical agents.

[0012] In certain embodiments, the cell expressing upregulated PI3K activity is a mutated endothelial cell, a tumor associated endothelial cell, or a tumor cell.

[0013] In certain embodiments, the mutated endothelial cell expresses elevated cell proliferation, elevated RAC1 activity or sprouting angiogenesis activity.

[0014] In certain embodiments, the cell is present in a vascular lesion.

[0015] In certain embodiments, the vascular lesion is caused by vascular malformations, a condition that results in malformation in vascular or lymphatic tissue or both.

[0016] In certain embodiments, the nanoparticle is a polymeric nanoparticle further comprising a core, wherein the core comprises the one or more pharmaceutical agents, and the one or more polymer layers surround the core.

[0017] In certain embodiments, the nanoparticle further comprises a core comprising the one or more pharmaceutical agents and poly-acrylic acid complexed with spermine, triethylamine, or Ca2+, or combinations thereof; and wherein the core is coated with the one or more polymer layers, wherein the polymer layers are each a PEG layer.

[0018] In certain embodiments, the pharmaceutical agent is selected from the group consisting of Rapamycin, Alpelisib, Trametinib, Binimetinib, Selumetinib, Dabrafenib, Cobimetinib, Torkinib and Palbociclib, or a combination thereof.

[0019] In certain embodiments, the pharmaceutical agent is Rapamycin or Alpelisib, or a combination of Rapamycin and Alpelisib.

[0020] In certain embodiments, the plurality of nanoparticles have an average diameter of from about 60 nm to about 210 nm.

[0021] In certain embodiments, the plurality of nanoparticles has a PDI of from about 0.150 to about 0.240.3LEGAL02 / 47570581V1Attorney Docket No. 035052 / 640791

[0022] In certain embodiments, the nanoparticle further comprises: a core comprising Rapamycin or Alpelisib or a combination thereof, and poly-acrylic acid complexed with spermine, triethylamine, or Ca2+, or combinations thereof; wherein the core is coated with the one or more polymer layers, wherein the polymer layers are each a PEG layer; and the plurality of nanoparticles have an average diameter of between about 70 nm and about 120 nm.

[0023] In certain embodiments, the nanoparticle formulation further comprises a pharmaceutically acceptable excipient.

[0024] In certain embodiments, the disease is selected from the group consisting of cancer, vascular malformations and lymphatic malformations.

[0025] Additional embodiments are also described herein.BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0026] Having thus described the invention in general terms, reference will now be made to the accompanying drawings, which are not necessarily drawn to scale.

[0027] Figure 1A-C illustrates a 3D culture of endothelial cells expressing constitutively active PIK3CA isoforms recapitulates irregular and enlarged vessel phenotypes associated with VMs. (A) Western blot of p-AKTl(Thr308), p-AKTl(Ser473) and p-S6K(Thr389) levels demonstrating elevated PI3K signaling as measured by increased phosphorylated-AKTl in cells expressing mutated variants of PIK3CA. (B) Control cells and endothelial cells expressing PIK3CA-activating mutations cultured in 3D fibrin matrices and imaged at 0 and 168 hours after seeding. (Scale bar = 1000 pm) (C) Confocal images (maximum intensity projections of the bottom half of vascular networks) of GFP and PIK3CAE542Kvascular networks labeled with fibrin, DAPI (nucleus), and actin. Vascular networks were fixed 3 days after seeding. (Scale bars = 100 pm).

[0028] Figure 2A-E illustrates inhibition of cell hyperproliferation alleviates the development of malformed vasculature by PIK3CA mutant endothelial cells. (A) Representative images of GFP, PIK3CAWT, PIK3CAE542K, and PIK3CAE545K cells stained for DAPI and EdU. Cells were pulse-labeled for 30 minutes with EdU prior to fixation. (Scale bar =100 pm) (B) Percentage of Edu positive labelled cells, (n = 4; mean ± s.e.m; One-way ANOVA with Tukey post-test, ns = not significant, **p < 0.01; ****p < 0.0001). (C) Representative images of DMSO treated and4LEGAL02 / 47570581V1Attorney Docket No. 035052 / 640791 mitomycin treated PIK3CA-mutant vascular networks. For mitotic inhibition, PIK3CAE342Kcells were pretreated with mitomycin-C (0.01 mg / ml) before culturing in fibrin hydrogel. (D-E) Cell count (D), volumetric quantification of ECM (E), of GFP control, DMSO treated PIK3CAE342K, and mitotically inhibited PJK3CAE542Kvascular networks.

[0029] Figure 3A-F illustrates Rapamycin and Alpelisib treatments improve and prevent the development of vascular malformation. (A) Representative images (maximum intensity projections) of PIK3CAE542Kvascular networks treated with DMSO, rapamycin (1 pM) or Alpelisib (2.5 pM) for 3 days. Vascular networks were labeled with fibrin, DAPI (nucleus), and actin. End points (marked as balls) were highlighted in 3D renderings of segmented vessels. Scale bars, 100 pm. (B to E) Quantification of cell number (B), ECM morphology (C), mean vessel length (D), and vessel branching (E) in GFP control, rapamycin-treated PIK3CAE542Kvascular networks, or Alpelisib-treated PIK3CAE542Kvascular networks (n > 3; means ± SEM; one-way ANOVA with Tukey post-test). (F) Western blot and quantification of relative p-AKTl(Thr308), p-AKTl(Ser473), p-S K^Thr389), and p-ERK l / 2(Thr202 / Tyr204) levels in PIK3CAE542Kcells treated with DMSO, rapamycin, or Alpelisib for 24 hours (n > 3; mean ± SEM; one-way ANOVA with Tukey post-test, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

[0030] Figure 4A-H illustrates treatment with trametinib (MEK1 / 2 inhibitor) and torkinib (mTORCl / 2 inhibitor) improves and rescues vascular phenotypes in PIK3CAE542Kvascular networks, respectively. (A) Western blot analysis of relative p-AKTl(Thr308), p-AKTl(Ser473), p- S6K(Thr389), and p-ERKl / 2(Thr202 / Tyr204) levels in PIK3CAE342Kcells treated with the indicated inhibitors. (B) Representative confocal images (maximum intensity projections) of PIK3CAE542Kvascular networks treated with DMSO, trametinib (MEK1 / 2 inhibitor, 1 pM) or torkinib (mTORCl / 2 inhibitor, 5 pM) for 3 days. Scale bars, 100 pm. (C to F) Quantification of relative p-AKTl(Thr308), p-AKTl(Ser473), p-S6K(Thr389), and p-ERKl / 2 levels in (A) (n > 3; means ± SEM; one-way ANOVA with Tukey post-test). (G and H) Quantification of matrix (G) and vessel branching morphology (H) in (B) (n > 3; mean ± SEM; one-way ANOVA with Tukey post-test, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

[0031] Figure 5A-B illustrate characterization of polymeric nanoparticles of different sizes. FNP was used to synthesize different formulations of polymeric nanoparticles with varying sizes (58 - 705 nm). Some formulations were labeled with a fluorescent probe to use in targeting experiments with endothelial cells. (A) DLS showing the scattering intensity distribution of 95LEGAL02 / 47570581V1Attorney Docket No. 035052 / 640791 formulations ranging from 58 to 705 nm and their respective polydispersity indices (PdTs). (B) TEM of formulation shown in dotted line in (A). Particles were adsorbed onto copper 400 mesh TEM grids were negatively stained with 2% uranyl acetate and imaged via TEM at 5000x magnification (scale bar = 5pm) and at 100,000x magnification (Scale bar = 200 nm).

[0032] Figure 6A-D illustrates the successful loading of Rapamycin into nanoparticles. (A-C) detection of Rapamycin by high-performance liquid chromatography coupled with a diode array detector (HPLC-DAD) at 277 nm, (A) blank consisting of the mobile phase, (B) elution of Rapamycin standard (75 pM) with a retention time of 8.1 min, and (C) elution of Rapamycin present in the nanoparticles. (D) Rapamycin calibration curve used to determine the drug concentration, the green datapoint indicates the NPs dilution used for the quantification.

[0033] Figure 7A illustrates cellular uptake of nanoparticles in healthy and PIK3CAE545Kmutant endothelial cells cultured in 2D. (A) To assess whether the expression PIK3CA activating mutation resulted in preferential nanoparticles endocytosis, we generated fluorescent Dil empty nanoparticles of different sizes (ranging from 61.29 nm to 206.80 nm) and compared the cellular uptake of nanoparticles in control and PIK3CAE543Kendothelial cells. (Scale bar = 100 pm).

[0034] Figure 8A-C illustrates preferential cellular uptake of nanoparticles by PIK3CAE545Kmutant endothelial cells cultured in 2D. (A) Cellular uptake was determined by quantifying the total Dil fluorescence intensity of each cell. Each data point represents data from a single cell, with n=3 independent experiments pooled, and mean ± standard deviation superimposed on individual data points (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 as determined by Kolmogorov- Smirnov test with Holm-Sidak multiple comparisons test). (B) Representative images of healthy HUVECs and PIK3CAE543KHUVECS treated with Dil-labeled nanoparticles independently prepared with cells dosed and uptake quantified blindly. Uptake of nanoparticles by cells as a function of nanoparticle diameter. The cellular uptake was determined by quantifying the total Dil fluorescence intensity within each cell using 3D computed cell surfaces to determine nanoparticles that were internalized vs. remaining on the cell surface. (C) Data were plotted as the integrated fluorescence intensity internal to the cell boundary per cell and are plotted as mean ± standard error. (N = 3-4 independent experiments, between 63 and 163 cells were counted per condition. *p<0.05, **p<0.01, as determined by Welch’s t-test for significance at each nanoparticle diameter. Scale bars = 40pm).6LEGAL02 / 47570581V1Attorney Docket No. 035052 / 640791

[0035] Figure 9A-B illustrates the effects of inhibition of endocytosis pathways on the uptake of nanoparticles. (A) Representative images of healthy and PIK3CAE545KHUVEC treated with endocytosis inhibitors Dynasore, EIP A, PitStop2 or DMSO load control after 8 hrs of treatment with 111 nm Dil-loaded nanoparticles. (B) Quantification of nanoparticle uptake with inhibitor treatment (n=3, ***p<0.001 as determined by one-way ANOVA with Tukey post test, scale bar = 40 pm).

[0036] Figure 10A-B illustrates preferential cellular uptake of nanoparticles by PIK3CAE545Kmutant endothelial cells cultured in 3D. (A-B) Uptake of 84.56 nm (A) and 111.30 nm (B) nanoparticles in 3D cell culture environment. To assess whether our findings in 2D studies is replicable in cells cultured in a 3D microenvironment, control and PZK3CAE345Kmutant endothelial cells cultured in 3D fibrin matrices were treated with 84.56 nm or 111.30 nm nanoparticles for 24 hours. (Scale bar = 10 pm).

[0037] Figure 11A-B illustrates release and effect of rapamycin from 110 nm nanoparticles after 24 hrs of treatment. (A) Viability of PIK3CAE545KHUVEC as determined by MTT assay after treatment of rapamycin encapsulated in 110 nm diameter nanoparticles vs. rapamycin in free solution. (B) Isolated cell viability for PIK3CAE545KHUVEC treated with rapamycin encapsulated in 110 nm diameter nanoparticles plotted on a logarithmic dosage scale.DETAILED DESCRIPTION

[0038] Described herein is the development of a nanomedicine strategy to specifically target cells harboring driving mutations for vascular and lymphatic malformations (VMs and LMs, respectively). The formulation consists of polymeric nanoparticles synthesized by Flash Nanoprecipitation (FNP) and / or inverse Flash Nanoprecipitation (iFNP) packaged with small molecule inhibitors that target downstream signaling components that drive the formation of microvascular lesions that underlie morbidity and mortality of patients with VMs and LMs. The treatment strategy is informed by mechanistic studies performed by the disclosing parties that identify a novel signaling mechanism in VM lesion formation (Aw et al. Science Advances 2023). The approach addresses a significant unmet need by selectively targeting cells harboring mutations, thus limiting broad toxicity observed with untargeted treatment. Preliminary data demonstrate selective uptake of nanoparticles by cells expressing driving mutations. Potential7LEGAL02 / 47570581V1Attorney Docket No. 035052 / 640791 treatment populations extend beyond VMs and LMS to other diseases and disorders caused by upregulated PIK3CA activity, including solid tumors.

[0039] VMs are caused by somatic mutations in genes involved in vascular development. The same genes are commonly implicated in malignant tumor angiogenesis. In particular, somatic activating mutations of PIK3CA have been identified to cause the majority (-80%) of cystic lymphatic malformations, and to drive generalized lymphatic anomalies, capillary malformations, and venous malformations. Histological and genetic analysis has identified that a minority of cells within lesions express the driving mutations.

[0040] Standard treatment of slow-flow malformations includes palliative care such as compression, analgesia, and coagulopathy correction, sclerotherapy or laser therapy for symptom relief and cosmetic purposes. However, these treatments are limited to superficial lesions, and repeated sclerotherapy is often required and can result in complications such as necrosis, pain, and peripheral neuropathy.

[0041] In clinical trials involving Rapamycin (sirolimus), a known mTORCl inhibitor, results indicated that while a complete cure was not achieved, 50% of patients experienced an improvement in quality of life, and 20% of patients saw a reduction in lesion size. Furthermore, dosing of rapamycin, especially for pediatric patients, remains challenging. More recently, Alpelisib (marketed as Vijoice), a selective inhibitor of the pl 10a subunit of PI3K, received approval for systemic therapy of advanced PROS patients. While effective in reducing the size of existing lesions, treatment with Alpelisib has been associated with severe reactions including diabetic ketoacidosis, hypertension, and severe cutaneous events in up to 35% of breast cancer patients with PIK3CA mutation. Additionally, a recent FDA advisory committee reported dosing challenges and side effects associated with inhibition of the PI3K pathway for blood malignancies. The limited indication of Alpelisib in systemic therapy and its associated side effects complicate the broader application of Alpelisib in VMs treatment.

[0042] PIK3CA activating mutations drive elevated Rael activity in endothelial cells, which drives lesion formation through dysregulated cytoskeletal dynamics. Rael regulates membrane ruffling and underlies internalization mechanisms. As described herein, the utilization of upregulated Rael activity in mutant cells represents an opportunity to specifically target mutant cells through increased uptake of NPs.8LEGAL02 / 47570581V1Attorney Docket No. 035052 / 640791

[0043] PIK3CA activating mutations drive elevated Rael activity in endothelial cells, which drives lesion formation through dysregulated cytoskeletal dynamics. Rael regulates membrane ruffling and underlies internalization mechanisms whereby upregulated Rael activity in mutant cells represents an opportunity to specifically target mutant cells through increased uptake of NPs. However, the targeting of the nanoparticle has been found to be particle diameter size-dependent. Data herein report on a microphy si ologi cal model of PIK3 CA -driven vascular malformations consisting of human umbilical vein endothelial cells (HUVECs) expressing PIK3CA activating mutations embedded in 3D hydrogels (Fig. 1-4). Mutant endothelial cells (ECs) overproliferate and develop into vascular networks characterized by complex, irregular, and enlarged lumens, consistent with clinical observations. Inhibition of mitosis and treatment with Rapamycin, Alpelisib, MEK1 / 2 inhibitor, or mTORCl / 2 inhibitor are effective in improving malformed vessel morphology (Fig. 2-4). Nanoparticles ranging from 58 nm to 705 nm in diameter (Fig 5) were tested by administering NPs encapsulated with Dil, a fluorescent probe to control HUVEC and PIK3CAE542KECS and quantifying cellular uptake 8 hours post administration. Cellular uptake of NPs ranging from 70 nm to 206 nm in diameter are more effectively internalized by PIK3CA- mutant ECs (Fig. 6-8). Described herein is size-dependent differential uptake by mutant cells as a mechanism to specifically target mutant cells to address current challenges for effective treatment due to toxicity and tolerability by patients. The resulting formulations involve advantageous therapeutic approaches for targeting dysfunctional endothelium and for targeting other cell types harboring mutations that drive hyperactive PI3K signaling, including solid tumors. Given the broad expression of the proteins targeted by current interventional approaches, increased specificity is necessary to reduce toxicity and improve patient outcomes.

[0044] Nano- and therapeutic delivery technologies possess the potential to present solutions for addressing challenges like inadequate therapeutic accumulation in vascular malformations and reducing side effects such as systemic toxicity. Among these technologies, polymeric nanoparticles synthesized through advanced methods like Flash Nanoprecipitation (FNP) and inverse Flash Nanoprecipitation (iFNP) offer a versatile platform for targeted delivery, allowing for specific targeting of cells within the vascular malformation with PIK3CA activating mutations. The encapsulation of therapies within nanoparticles can impart tailored properties, altering their behavior in vivo. FNP and iFNP uniquely tackle these challenges. These processes involve the rapid mixing of liquid streams containing polymers and therapies in specialized devices like micro9LEGAL02 / 47570581V1Attorney Docket No. 035052 / 640791 inlet vortex mixers (MIVMs) or confined impinging jet mixers (CIJs). This rapid mixing results in the consistent generation of homogeneous nanoparticles, with size and therapeutic loading finely tunable by adjusting the polymer-to-therapeutic ratios in the liquid streams.

[0045] As described herein, particular nanoparticle size ranges can provide specificity for targeting and delivery of therapeutics to cells harboring driving mutations and reduce delivery to healthy, neighboring cells.

[0046] The presently disclosed subject matter will now be described more fully hereinafter. However, many modifications and other embodiments of the presently disclosed subject matter set forth herein will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented in the foregoing descriptions. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. In other words, the subject matter described herein covers all alternatives, modifications, and equivalents. In the event that one or more of the incorporated literature, patents, and similar materials differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in this field. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.I. Definitions

[0047] The terms “subject,” “patient,” or “individual,” as used herein, are used interchangeably and refers to any animal, including mammals such as mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, primates, and humans. In some embodiments, the term refers to a subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired or needed. In some embodiments, the subject is a human. In some embodiments, the subject has experienced and / or exhibited at least one symptom of the disease, disorder, or condition to be treated and / or prevented. In some embodiments, the subject is suffering with from a vascular malformation, or a disease or condition characterized by a vascular malformation10LEGAL02 / 47570581V1Attorney Docket No. 035052 / 640791 including capillary (CM), lymphatic (LM), venous (VM), and arteriovenous (AVM), and combinations thereof.

[0048] As used herein, the term “vascular malformation” refers to a vascular anomaly where instead of normal, smooth vessels, the vessels enlarge or form tangles, pockets or shunting vessels that cause abnormal blood flow. A “vascular lesion” is a type of malformation.

[0049] The term “in need thereof’ would be a subject known or suspected of having or being at risk of a vascular malformation or a disease or condition characterized by a vascular malformation including capillary (CM), lymphatic (LM), venous (VM), cerebral cavernous malformations (CCM), and arteriovenous (AVM), and combinations thereof.

[0050] A subject in need of treatment would be one that has already developed the disease or condition. A subject in need of prevention would be one with risk factors of the disease or condition.

[0051] The terms “treat,” “treatment” and the like refer to a means to slow down, relieve, ameliorate or alleviate at least one of the symptoms of the disease, or reverse the disease after its onset.

[0052] The terms “prevent,” “prevention” and the like refer to acting prior to overt disease onset, to prevent the disease from developing or minimize the extent of the disease or slow its course of development.

[0053] As used herein, the term “mutated endothelial cell” refers to an endothelial cell harboring a mutation in its DNA. An endothelial cell is a specialized type of cell that forms the inner lining of blood vessels and lymphatic vessels, creating a single-cell layer called the endothelium. These cells play a crucial role in regulating blood flow, fluid exchange between the blood and tissues, and maintaining vascular health by controlling blood pressure and platelet aggregation. A “tumor associated endothelial cell” refers to a cell lines the blood vessels of tumors and controls the flow of nutrients to the surrounding tumor tissue.

[0054] As used herein, the term “elevated Rael activity” means any detectable increase in Rael levels above normal expression.

[0055] As used herein, the term “PIK3CA” refers to a gene that encodes a subunit of an enzyme called phosphatidylinositol 3-kinase. Phosphoinositide 3-kinases (PI3Ks), also called phosphatidylinositol 3-kinases, are a family of enzymes involved in cellular functions such as cell growth, proliferation, differentiation, motility, survival and intracellular trafficking, which11LEGAL02 / 47570581V1Attorney Docket No. 035052 / 640791 in turn are involved in cancer. Phosphatidylinositol 3-kinase is composed of an 85 kDa regulatory subunit and a 110 kDa catalytic subunit. PIK3CA, which encodes the pl 10a, is frequently mutated in cancer. Non-limiting examples of PI3Ka inhibitors include compounds, molecules, chemicals, polypeptides and proteins that inhibit and / or reduce the expression and / or activity of PI3KCA. A non-limiting example of PI3KCA inhibitors is Alpelisib.

[0056] As used herein, the term “MEK1 / 2” refers to a dual-specificity protein kinase that phosphorylates tyrosine and threonine residues in ERK1 / 2, which activates ERK1 / 2. ERK1 / 2 are protein-serine / threonine kinases that have many cytosolic and nuclear substrates. Mitogen- activated protein kinase (MAPK) cascades are key signaling pathways involved in the regulation of normal cell proliferation, survival, and differentiation. Aberrant regulation of MAPK cascades contribute to cancer and other human diseases. In particular, the extracellular signal-regulated kinase (ERK) MAPK pathway has been implicated in many cancers as a therapeutic target. ERK is a downstream component of an evolutionarily conserved signaling module that is activated by the Raf serine / threonine kinases. Raf activates the MAPK / ERK kinase (MEK) 1 / 2 dual-specificity protein kinases, which then activate ERK1 / 2. The mutational activation of Raf in human cancers supports the important role of this pathway in human oncogenesis. Additionally, the Raf-MEK- ERK pathway is a key downstream effector of the Ras small GTPase, the most frequently mutated oncogene in human cancers. Lastly, Ras is a key downstream effector of the epidermal growth factor receptor (EGFR), which is mutationally activated and / or overexpressed in a wide variety of human cancers. ERK activation also promotes upregulated expression of EGFR ligands, promoting an autocrine growth loop critical for tumor growth. Thus, certain embodiments are directed to modulating (e.g., inhibiting) the EGFR-Ras-Raf-MEK-ERK signaling pathway, for example, by inhibiting the MEK 1 / 2 dual-specificity protein kinases. Some embodiments therefore include small molecules that inhibit MEK1 and / or MEK2. Non-limiting examples of small molecule MEK 1 / 2 inhibitors include trametinib, selumetinib, cobimetinib, and binimetinib. Additional examples ofMEKl and MEK2 inhibitors are described, for example, in U.S. Application No. 2009 / 0124595; U.S. Application No. 2009 / 0246198; and U.S. Application No. 2010 / 0004234, which are incorporated by reference.

[0057] As used herein, the term “CDK 4 / 6” refers to cyclin-dependent kinases (CDKs) that drive cell division. Of particular importance to the cancer field are D-cyclins, which bind and activate CDK4 and CDK6. Cyclin-dependent kinases (CDKs) are a family of protein kinases that12LEGAL02 / 47570581V1Attorney Docket No. 035052 / 640791 play a role in regulating the cell cycle. CDK4 and CDK6 regulate the cell G1 phase progression and the Gl / S transition of the cell cycle. CDK4 and CDK6 have been shown to phosphorylate and thus regulate the activity of the tumor suppressor Retinoblastoma protein, and are overexpressed or otherwise unbalanced in a variety of tumors. For example, the overexpression of CDK4 and CDK6 can provide cancer cells certain hallmarks of cancer, including deregulation of the cell cycle and of cell metabolism. Thus, certain embodiments are directed to modulating (e.g., inhibiting) CDK 4 / 6-regulated pathways, for example, by inhibiting the CDK4 and / or CDK6 kinases. Included are agents that inhibit the ability or activity of CDK 4 / 6 to phosphorylate a serine or threonine residue on proteins, or inhibit the interaction of CDK 4 / 6 with other proteins / ligands that are involved in the signal pathway. CDK4 and CDK6 form a complex with Cyclin D to regulate cell cycle progression from G1 to S phase. CDK4 has been shown to interact with retinoblastoma (Rb), CDC37, CDKN1B, CDKN2B, CDKN2C, CEBPA, CCND1, CCND3, DBNL, MyoD, P16, PCNA, and SERTAD1. CDK6 has been shown to interact with retinoblastoma (RB), CDKN2C, PPM1B, Cyclin D3, Cyclin DI, and PPP2CA. Thus, in certain embodiments, the CDK 4 / 6 inhibitor reduces the interaction of CDK4 and / or CDK6 with any one or more of the foregoing proteins or ligands. Some embodiments therefore include small molecules that inhibit CDK4 and / or CDK6. Non-limiting examples of small molecule CDK 4 / 6 inhibitors include ribociclib, abemaciclib, and palbociclib. Additional examples of CDK 4 / 6 inhibitors are described, for example, in WO 2007 / 140222; WO 2010 / 020675; and U.S. Application No. 2013 / 0035336, which are incorporated by reference.

[0058] As used herein, the phrase “mTORCl / 2” or “mTORC 1 / 2 inhibitor” refers to a catalytic mTOR inhibitor that interacts with and reduces the kinase activity of both mTORC 1 and mT0RC2 complexes. The term “mTORC 1 / 2”, as used herein, refers to the mTORC 1 complex, the mT0RC2 complex, or both. In some embodiments of the methods of the invention, the mTOR inhibitor binds to and directly inhibits both mTORC 1 and mT0RC2.

[0059] As used herein, the term “upregulated PI3K activity” refers to any aberrant activation of the phosphoinositide pathway, in particular, by a mutation in a cell.

[0060] As used herein, the term “plurality of nanoparticles” and the like refer to two or more particles having a shape with a diameter wherein the nanoparticles in the plurality have an average diameter between about 60 nm to 200 nm. Particle size can be determined using any method known in the art, including, but not limited to, sedimentation field flow fractionation, photon correlation13LEGAL02 / 47570581V1Attorney Docket No. 035052 / 640791 spectroscopy, disk centrifugation, and dynamic light scattering (using, for example, a submicron particle sizer such as the NICOMP particle sizing system from AutodilutePAT Model 370; Santa Barbara, CA).

[0061] As used herein, the term “Flash Nanoprecipitation” refers to a process involving supersaturation needed for particle nucleation by fast mixing of a stream containing a dissolved solute and a stabilizing molecule with an opposing stream containing a miscible solvent, which acts as a non-solvent for the solute and stabilizer. Turbulent mixing occurs in a confined volume, which provides the supersaturation conditions required for simultaneous precipitation of the solute and stabilizer. Flash Nanoprecipitation has been used to encapsulate poorly water-soluble pharmacologies. The term “Inverse Flash Nanoprecipitation” refers to a process that unlike a traditional FNP process, the inverse process is used to encapsulate highly water-soluble drugs, whereby the nanoparticle has hydrophobic ends pointing out and hydrophilic tails pointing inward. Inverse Flash Nanoprecipitation is suitable for encapsulating Alpelisib.

[0062] As used herein, the term “physiological conditions” refers to the range of conditions of temperature, pH, and tonicity (or osmolality) normally encountered within tissues in the body of a living human.

[0063] The term “w vitro11refers to artificial environments and to processes or reactions that occur within an artificial environment (e.g., a test tube).

[0064] The term “in vivo11refers to natural environments (e.g., a cell or organism or body) and to processes or reactions that occur within a natural environment, such as the human body.

[0065] The terms “effective amount” or “therapeutically effective amount” refer to a sufficient amount of the composition to provide the desired biological result. That result can be reduction and / or alleviation of the signs, symptoms, or causes of a disease or medical condition, or any other desired alteration of a biological system. For example, an “effective amount” for therapeutic use is the amount of a composition that is required to provide a clinically relevant change in a disease state, symptom, or medical condition. An appropriate “effective” amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation. Thus, the expression “effective amount” generally refers to the quantity for which the active substance has a therapeutically desired effect. Effective amounts or doses of the compositions of the embodiments may be ascertained by routine methods, such as modeling, dose escalation, or clinical trials, taking into account routine factors, e.g., the mode or route of administration or drug14LEGAL02 / 47570581V1Attorney Docket No. 035052 / 640791 delivery, the pharmacokinetics of the agent, the severity and course of the infection, the subject's health status, condition, and weight, and the judgment of the treating physician. An exemplary dose is in the range of about 1 pg to 2 mg of active agent per kilogram of subject's body weight per day. The total dosage may be given in single or divided dosage units (e.g., BID, TID, QID). Once improvement of the patient's disease has occurred, the dose may be adjusted for preventative or maintenance treatment. For example, the dosage or the frequency of administration, or both, may be reduced as a function of the symptoms, to a level at which the desired therapeutic or prophylactic effect is maintained. Of course, if symptoms have been alleviated to an appropriate level, treatment may cease. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of symptoms. Patients may also require chronic treatment on a longterm basis.

[0066] The term “excipient” or “pharmaceutically acceptable excipient” means a pharmaceutically-acceptable material, composition, or vehicle, such as a liquid or solid fdler, diluent, carrier, solvent, or encapsulating material. In some embodiments, each component is “pharmaceutically acceptable” in the sense of being compatible with the other ingredients of a pharmaceutical formulation, and suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit / risk ratio. See, e.g., Remington: The Science and Practice of Pharmacy, 21st ed.; Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 6th ed.; Rowe et al., Eds.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009; Handbook of Pharmaceutical Additives, 3rd ed.; Ash and Ash Eds.; Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2nd ed.; Gibson Ed.; CRC Press LLC: Boca Raton, FL, 2009.

[0067] The term “pharmaceutically acceptable” as used herein indicates that the compound, or salt or composition thereof is compatible chemically and / or toxicologically with the other ingredients comprising a formulation and / or the subject being treated therewith.

[0068] The term “administration” or “administering” refers to a method of giving a dosage of a nanoparticle or nanoparticle formulation to a subject. The method of administration can vary depending on various factors, e.g., the components of the pharmaceutical composition, the site of the disease, and the severity of the disease.15LEGAL02 / 47570581V1Attorney Docket No. 035052 / 640791

[0069] Additional definitions may be provided below.II. Therapeutic methods

[0070] In certain embodiments, the subject matter described herein is directed to a method of selectively targeting a cell expressing upregulated PI3K activity in a subject in vivo, the method comprising: administering to the subject a plurality of nanoparticles having an average diameter from about 50 nm to about 220 nm, the nanoparticles comprising: one or more polymer layers; and, one or more pharmaceutical agents.

[0001] In certain embodiments, the subject matter described herein is directed to a method of treating a disease associated with upregulated PI3K activity in a subject, the method comprising: administering to the subject a therapeutically effective amount of a pharmaceutical agent in a nanoparticle formulation, wherein the nanoparticle formulation comprises a plurality of nanoparticles having an average diameter of from about 50 nm to about 220 nm, and wherein the nanoparticles comprise a polymer and one or more pharmaceutical agents.

[0002] In certain of the above embodiments, the cell expressing upregulated PI3K activity is a mutated endothelial cell, a tumor associated endothelial cell, or a tumor cell.

[0003] In certain of the above embodiments, the cell expressing upregulated PI3K activity is an endothelial cell with PIK3CA mutation or TEK mutation.

[0004] In certain of the above embodiments, the mutated endothelial cell expresses elevated cell proliferation, elevated RAC1 activity or sprouting angiogenesis activity.

[0005] In certain of the above embodiments, the nanoparticle comprises a poly-acrylic acid. In certain embodiments, the poly-acrylic acid can be complexed with spermine and / or triethylamine and / or Ca+2. In some aspects, the nanoparticle comprises of a poly (acid) (e.g. poly (aspartic acid), poly(glutamic acid), poly(itaconic acid), poly(malic acid)) complexed with small molecule polyamines (e.g. spermine, putrescine, cadaverine) or polymer-based poly(amine) (e.g. poly (e thy lenimine, poly(propylamine), poly(lysine), poly(arginine) poly(histidine)). In certain of the above embodiments, the nanoparticle comprises a core, wherein the core comprises the polymer.16LEGAL02 / 47570581V1Attorney Docket No. 035052 / 640791

[0006] In certain of the above embodiments, the polymer is coated with one or more layers of polymers. In certain embodiments, the coating on the core comprises a hydrophobic polymer layer (ex. PLA, poly(glycolic acid), poly(lactic-co-gly colic acid), poly(caprolactone), poly (propylene fumarate), poly(ortho-esters), poly(caprolactam), poly(anhydrides)). Still another layer can be present directly on top of the core or on top of the polymer coating that is adjacent to the core. This layer can be useful as a protective layer. This layer comprises a polymer selected from the group consisting of PEG, poly(2-ethyl- 2-oxazoline), poly(carboxybetaine), poly(sulfobetaine), poly(vinyl alcohol), poly(vinylpyrrolidone), and hyaluronic acid. Additionally, in some embodiments, the nanoparticle comprises dual encapsulated active agents, optionally a biologic and a small molecule.

[0007] In certain of the above embodiments, the cell is present in a vascular lesion.

[0008] In certain of the above embodiments, the vascular lesion is caused by vascular malformations, a condition that results in malformation in vascular or lymphatic tissue or both.

[0009] In certain of the above embodiments, the malformation is present in one or more of a vein, an artery, a capillary, or a lymphatic vessel.

[0010] In certain of the above embodiments, the nanoparticle is a polymeric nanoparticle further comprising a core, wherein the core comprises the one or more pharmaceutical agents, and the one or more polymer layers surround the core.

[0011] In certain of the above embodiments, the one, two or three polymer layers surround the core.

[0012] In certain of the above embodiments, the one or more polymer layers surrounding the core can comprise polyethylene glycol (PEG).

[0013] In certain of the above embodiments, the agent is a precipitated hydrophobic drug, and the one or more polymer layers surrounding the core can comprise a amphiphilic diblock copolymer.

[0014] In certain of the above embodiments, the amphiphilic diblock co-polymer comprises a hydrophobic block, wherein the hydrophobic block comprises polylactide.

[0015] In certain of the above embodiments, the agent is a precipitated hydrophobic drug, and the one or more polymer layers surrounding the core comprises an amphiphilic triblock copolymer.17LEGAL02 / 47570581V1Attorney Docket No. 035052 / 640791

[0016] In certain of the above embodiments, the amphiphilic triblock co-polymer comprises a hydrophobic block, wherein the hydrophobic block comprises polypropylene oxide.

[0017] In certain of the above embodiments, the amphiphilic triblock co-polymer comprises a hydrophobic block, wherein the hydrophobic block comprises polyethylene oxide.

[0018] In certain of the above embodiments, the nanoparticle can further comprise a core comprising the one or more pharmaceutical agents and poly-acrylic acid complexed with spermine, triethylamine, or Ca2+, or combinations thereof; and wherein the core is coated with the one or more polymer layers, wherein the polymer layers are each a PEG layer.

[0019] Useful pharmaceutical agents include small molecule inhibitors of target enzymes, complexes and the like. An inhibitor can have an IC50 value of about 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 50 nM or less, 10 nM or less, or 1 nM or less for the target, as ascertained in an in vitro assay. In certain embodiments, the inhibitor exhibits selectivity for, or alternatively inhibits one or more, targets of the same family or isoforms, such as mTORCl and mT0RC2. In any of the above embodiments, the agent can be a precipitated hydrophobic drug.

[0020] In certain of the above embodiments, the pharmaceutical agent can be a PIK3CA inhibitor, mTORCl inhibitor, mTORC2 inhibitor, mTORCl / 2 inhibitor, MEK inhibitors, or CDK4 / 6 inhibitor, or a combination of any two of the pharmaceutical agents.

[0021] In certain of the above embodiments, the pharmaceutical agent can be selected from the group consisting of Rapamycin, Alpelisib, Trametinib, Binimetinib, Selumetinib, Dabrafenib, Cobimetinib, Torkinib and Palbociclib, or a combination thereof.

[0022] In certain of the above embodiments, the pharmaceutical agent can be Rapamycin or Alpelisib, or a combination of Rapamycin and Alpelisib.

[0023] In certain of the above embodiments, the pharmaceutical agent can be Rapamycin.

[0024] In certain of the above embodiments, the nanoparticle can comprise two pharmaceutical agents, wherein the agents are dual encapsulated active agents or are separate.

[0025] In certain of the above embodiments, the plurality of nanoparticles can have an average diameter of from about 60 nm to about 210 nm.

[0026] In certain of the above embodiments, the plurality of nanoparticles can have an average diameter of about 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 or 210 nm.18LEGAL02 / 47570581V1Attorney Docket No. 035052 / 640791In certain of the above embodiments, the plurality of nanoparticles can have an average diameter of about 110 nm ± 20 nm.

[0027] In certain of the above embodiments, the plurality of nanoparticles can have a size as described herein and a PDI of from about 0.150 to about 0.240.

[0028] In certain of the above embodiments, the nanoparticles of the plurality are preferentially taken up by the cell with upregulated PI3K activity.

[0029] In certain of the above embodiments, the nanoparticle can further comprise: a core comprising Rapamycin or Alpelisib or a combination thereof, and poly-acrylic acid complexed with spermine, triethylamine, or Ca2+, or combinations thereof; wherein the core is coated with the one or more polymer layers, wherein the polymer layers are each a PEG layer; and the plurality of nanoparticles have an average diameter of between about 70 nm and about 120 nm.

[0030] As described herein, particular nanoparticle size ranges can provide specificity for targeting and delivery of therapeutics to cells harboring driving mutations and reduce delivery to healthy, neighboring cells. The plurality of nanoparticles have a size range that has been shown to exhibit preferential uptake in target cells. In any of the above embodiments, the nanoparticles do not comprise a targeting moiety.

[0031] In certain of the above embodiments, the nanoparticle formulation can further comprise a pharmaceutically acceptable excipient.

[0032] In certain of the above embodiments, disease is selected from the group consisting of cancer, vascular malformations and lymphatic malformations.

[0033] In certain of the above embodiments, disease is selected from the group consisting of vascular malformations and lymphatic malformations.

[0034] In certain of the above embodiments, upregulated PI3K activity is a result of PIK3CA, TEK, KRAS, NRAS, or BRAF activating mutations in endothelial cells.

[0035] In certain of the above embodiments, the nanoparticles of the plurality are prepared by Flash Nanoprecipitation (FNP) and / or inverse Flash Nanoprecipitation (iFNP).

[0036] The nanoparticle formulations may be administered in either single or multiple doses. The nanoparticle formulations may be administered by various methods including, for example, rectal, buccal, intranasal and transdermal routes. In certain embodiments, the nanoparticle formulations may be administered by intralesional injection, intra-arterial injection or19LEGAL02 / 47570581V1Attorney Docket No. 035052 / 640791 intravenously. One mode for administration is parenteral, for example, by injection. The forms in which the nanoparticle formulations described herein may be incorporated for administration by injection include, for example, aqueous or oil suspensions, or emulsions, with sesame oil, corn oil, cottonseed oil, or peanut oil, as well as elixirs, mannitol, dextrose, or a sterile aqueous solution, and similar pharmaceutical vehicles. In certain embodiments, the formulation is contained in a prefdled syringe of appropriate size and as is known in the art for storing an injectable formulation such as those described herein. In certain embodiments, a prefdled syringe comprises a formulation s a plurality of nanoparticles having an average diameter from about 50 nm to about 220 nm, or the plurality of nanoparticles can have an average diameter of from about 60 nm to about 210 nm. In certain of the above embodiments, the plurality of nanoparticles in a prefdled syringe can have an average diameter of about 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 or 210 nm. In certain of the above embodiments, the plurality of nanoparticles in a prefdled syringe can have an average diameter of about 110 nm ± 20 nm. In certain of the above embodiments, the plurality of nanoparticles in a prefdled syringe can have a size as described herein and a PDI of from about 0.150 to about 0.240.

[0037] The specific dose level of a nanoparticle or nanoparticle formulation described herein for any particular subject is within the purview of a trained clinician.

[0038] Furthermore, it is to be understood that in this written description support for actual numerical values is provided even when a range is provided. Disclosed ranges encompass all discrete values within that range, regardless of whether such discrete values are explicitly specified.III. Methods of Making a Plurality of Nanoparticles

[0039] In an embodiment, described herein is a method of preparing a plurality of nanoparticles comprising an encapsulated active agent or agents, the method comprising a Flash Nanoprecipitation (FNP) process comprising mixing two solvent streams, wherein a first solvent stream is an aqueous solvent, and a second solvent stream comprises an organic solvent and polymers; collecting a nanoparticle suspension; and, selecting a diameter size of nanoparticles to prepare a plurality of nanoparticles having an average diameter from about 60 nm to about 220 nm.20LEGAL02 / 47570581V1Attorney Docket No. 035052 / 640791

[0040] The disclosed subject mater is further described in the following non-limiting Examples. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only.EXAMPLESMaterial and Methods

[0041] Generation of endothelial cells expressing PIK3CA activating mutations: HUVECs (Lonza) were cultured in growth medium (EGM-2, Lonza). HLFs (Lonza) were cultured in growth medium (FGM-2, Lonza), and human embryonic kidney (HEK)-293T cells (Clonetech) were grown in high-glucose Dulbecco’s modified Eagle’s medium (Hyclone) supplemented with 10% fetal bovine serum (Hyclone) and 1% penicillin / streptomycin (Life Technologies). All cells were cultured at 37°C and 5% CO2 in a humidified incubator. Plasmids encoding pHAGE-EFlaL- eGFP-W (Addgene, plasmid #126686, a gift from D. Koton), pHAGE -PIK3CA-E542K-ires-GFP, pHAGE-PIK3CA-E545K-ires-GFP, pHAGE-PIK3CA-H1047R (Addgene, plasmid nos. 116479, 116485, and 116500 gifts from G. Mills and K. Scott), and pHAGE-PIK3CA-WT-ires-GFP were used for lentivirus generation. Q5 site-directed mutagenesis (New England Biolabs) was performed on pHAGE-PIK3CA-E542K-ires-GFP to generate pHAGE-PIK3CA-WT-ires-GFP. For lentivirus production, HEK-293T cells were transfected with pHAGE-plasmid, pSPAX2 plasmid, and pMD2.G plasmid (Addgene, plasmid nos. 12260 and 12259, gifts from D. Trono) using calcium phosphate transfection. Lentiviral supernatants were collected 2 days after transfection and concentrated using PEG-IT (System Biosciences, Palo Alto, CA) viral precipitator. Concentrated lentivirus was resuspended in phosphate-buffered saline (PBS). Serial dilutions of lentiviral particles were performed to determine the optimal concentration for transduction. Concentrated lentiviruses were subsequently added to 100,000 HUVECs cultured in a six-well plate. Transduction efficiency of GFP constructs was determined by GFP immunofluorescence. HUVECs transduced with pHAGE-PIK3CA-H1047R were selected with 2 pg / mL puromycin in EGM-2 media for 2 passages. Transduced HUVECs were used between passages 5 and 13.

[0042] Hydrogel formation, seeding in PDMS wells, immunostaining and imaging of microvascular networks: Fresh solutions of fibrinogen from bovine plasma (Millipore Sigma) were prepared before each seeding by dissolving fibrinogen powder in Dulbecco’s phosphate-21LEGAL02 / 47570581V1Attorney Docket No. 035052 / 640791 buffered saline (DPBS; Millipore Sigma) to a concentration of 5 mg / ml and incubating at 37°C for at least 30 min before filter sterilization with a 0.2-pm syringe filter (Millipore Sigma). Thrombin from bovine plasma was solubilized in DPBS to a concentration of 100 U / ml, aliquoted, frozen at -80°C, and thawed immediately before each seeding. HUVECs and HLFs were lifted from culture flasks with 0.25% (w / v) trypsin-EDTA (Thermo Fisher Scientific), centrifuged at 200g for 5 min. HUVECs were resuspended at 13 * 106cells / ml of EGM-2. For each well, 50 pl of cells in fibrin prepolymer were prepared by mixing 25 pl of fibrinogen (5 mg / ml), 11.5 pl of 13 x 106HUVECs / ml EGM-2, 13 pl of EGM-2, and 0.5 pl of thrombin (100 U / ml) for final cell concentrations of 3 * 106cells / ml in the total slurry. To visualize ECM degradation and vascular lesion formation, 1.5 mg / ml of Alexa Fluor 647-conjugated fibrinogen (Thermo Fisher Scientific) was added to the hydrogel slurry. After mixing with a pipette to avoid bubbles, a 50-pl slurry was added to each PDMS well, incubated for 15 min at 37°C in a cell culture incubator, with inversion every few minutes to allow cells to be distributed in 3D. Media were changed daily and microvascular network within devices were fixed 3 days after seeding. For mitotic inhibition, HUVECs plated on 10-cm dishes were incubated with mitomycin-C (0.01 mg / mL; Selleckchem) for 2.5 hours at 37°C. Mitomycin-C-treated HUVECs were trypsinized and seeded into microfluidic devices as described above. To observe network assembly over time, PDMS wells loaded with HUVECs expressing GFP were imaged daily with a laser scanning confocal microscope (FV3000, Olympus) with a 488-nm laser diode. Z-stacks of the gel volume were taken at xio magnification [U Plan S-Apo, 0.4-numerical aperture (NA) air objective, Olympus], For end point immunostaining, PDMS wells or devices were fixed with 4% paraformaldehyde (Millipore Sigma- Aldrich) in PBS containing calcium and magnesium (PBS++) at 37°C for 15 min. After rinsing twice with PBS++, devices were left on a laboratory rocker for 24 hours in PBS++ to wash. Cells were then permeabilized with 0.3% Triton X-100 (Millipore Sigma) for 10 min at RT, and nonspecific antibody binding was blocked with 2% (w / v) bovine serum albumin (BSA) in PBS++ for 24 hours at RT. Wells and devices were then sealed in tissue culture plates and kept at 4°C until antibodies were added. Samples cultured in microfluidic devices were imaged daily at *4 magnification (U Plan Fluor, 0.13-NA air objective, Olympus) on a wide-field microscope and fixed 3 days after seeding. For immunostaining, cells were permeabilized with 0.3% Triton X-100. F-actin was labeled with Alexa Fluor 488 or rhodamine-phalloidin (Thermo Fisher Scientific), and the nucleus was labeled with DAPI (Thermo Fisher Scientific) diluted in22LEGAL02 / 47570581V1Attorney Docket No. 035052 / 640791PBS++ (1 :200 and 1 : 1000, v / v, respectively) for 1 hour at RT before rinsing three times over 30 min with PBS++. Images were acquired with an Olympus FV3000 laser scanning confocal microscope with a 30* U Plan S-Apo N 1.05-NA silicone oil immersion objective, and images were adjusted for brightness and contrast using ImageJ.

[0043] Quantification of vascular network topology and fibrin void regions: Confocal z- stacks (4-pm step size, captured from top to bottom of the HUVEC channel within microfluidic devices) were used for measurement of vascular network topology. Rolling z projections (average over five z sections, Fiji) were performed to increase the signal-to-noise ratio in the images. Maximal intensity rolling z projections were performed for DAPI and actin channels, and minimum intensity rolling z projection was performed for the fibrin ECM channel. Contrast enhancement (0.35% of saturated pixels) was used to normalize image brightness across the z- stack. The processed z-stacks were exported as image sequences for 2D segmentation in CellProfiler. DAPI and actin channels were used for segmenting vascular networks. An adaptive minimum cross-entropy thresholding method (size of adaptive window = 250 pixels) was used for segmenting vascular networks. Briefly, a binary mask containing segmented nuclei was used as a reference for segmenting vascular networks labeled with actin. Binarized images were reconstructed into a z-stack and used as input images for 3D skeletonization, pruning, and vasculature analysis in VesselVio. To quantify fibrin void regions, processed fibrin images were binarized using adaptive thresholding (size of adaptive window = 100 pixels, Sauvola thresholding method). Binarized images were inverted and reassembled into z-stacks. Volume filling and 3D void volume analysis of binarized fibrin void regions was performed in VesselVio. Because of inaccuracies in skeletonization algorithm in extracting topologies of cystic and dilated mutant vascular networks, the branch and end points computations of mutant networks treated with dimethyl sulfoxide condition were not included in statistical comparisons between treatments.

[0044] Western blot assays: Cells were cultured in complete media unless otherwise noted. For measuring relative changes in phospho-AKT, phospho-S6K, and phospho-ERK level in response to inhibitors, HUVECs were cultured in media with inhibitors for 24 hours. Rapamycin, alpelisib (BYL719), Y-27362 2HC1, trametinib (GSK1120212), batimastat (BB-94), EHT1864 2HC1, and PF-3758309 were from Selleckchem. Cells were lysed on ice with radioimmunoprecipitation assay buffer (Thermo Fisher Scientific) containing Halt protease and phosphatase inhibitor (Thermo Fisher Scientific). Clarified lysates were resolved on Novex 4 to23LEGAL02 / 47570581V1Attorney Docket No. 035052 / 64079112% bis-tris gel (Thermo Fisher Scientific) and transferred to a polyvinylidene difluoride membrane. Standard immunoblotting protocols were performed for Western blotting with primary antibodies used at the following concentrations: anti-pl 10a (1 : 1000; Cell Signaling Technology, #4249), anti-p-AKT-Thr308(1 : 1000, Cell Signaling Technology, #9275), anti-p-AKT- Ser473(1 : 1000; Cell Signaling Technology, #9271), anti-AKT (1: 1000; Cell Signaling Technology, #9272), anti-pSOK-Thr389(1 : 1000; Cell Signaling Technology, #9205), anti-S6K (1: 1000; Cell Signaling Technology, #9202), anti-pERKl / 2-Thr202 / Tyr204(1 :1000; Cell Signaling Technology, #9101), anti-ERKl / 2 (1: 1000; Cell Signaling Technology), and anti-glyceraldehyde- 3-phosphate dehydrogenase (1 :2000; Cell Signaling Technology, #2118). Horseradish peroxidase-conjugated secondary antibodies and SuperSignal West Femto or Clarity Western ECL chemiluminescent substrate were used for detection. Western blot images were quantified with Fiji / ImageJ.

[0045] Edu Assays: For EdU assays, cells were pulse-labeled with 10 pM EdU for 30 min before fixation and immunostaining. Click-iT EdU Cell Proliferation assay (Thermo Fisher Scientific) was performed according to the manufacturer’s instructions.Example 1 : Nanoparticle Preparation

[0046] Generating fluorescent empty nanoparticles of different sizes: Poly(D,L-lactide)- PEG-methyl ether (PDLLA-MPEG) (5k-5k, Nanosoft Polymers, 9511-5000-5000), Resomer® R 202 H Poly(D,L-lactide) (PDLLA) (Sigma Aldrich, 719978), THF (Fisher Scientific, T425-4), Dil (Invitrogen, D3911). PDLLA-MPEG 5k-5k and PDLLA were dissolved in THF at 100 mg / mL. Dil fluorescent tracer was dissolved in DMSO at 15 mg / mL. The following formulations were prepared to generate fluorescently labeled Dil loaded nanoparticles of different sizes.

[0047] Table 1. Synthesis of fluorescently labelled nanoparticles. TMC refers to the total mass concentration of the syringe volume. Core % refers to the percent concentration of PDLLA within the hydrophobic nanoparticle core.Table 1.24LEGAL02 / 47570581V1Attorney Docket No. 035052 / 640791

[0048] Polymeric NPs were synthesized via flash nanoprecipitation (FNP) with a confined impinging jet mixer (CIJ). MilliQ water was used as the aqueous solvent. Briefly, a CIJ was fitted with two 1 mL syringes where one syringe containing 500 pL of organic solvent (PDLLA + PDLLA-MPEG (5k-5k) + Dil dissolved in THF) and another syringe containing 540 uL of aqueous solvent (MilliQ water). The two solvent streams were mixed together using the CIJ and were collected in a scintillation vial containing 4 mL of MilliQ water. The collected nanoparticles suspensions were mixed for 5 minutes at room temperature using a magnetic stirrer. The suspensions were dialyzed overnight in MiliQ water at 4°C. After dialysis, nanoparticles were stored at 4°C.

[0049] Nanoparticle size measurements with Dynamic Light Scattering: Measurements for nanoparticle size with intensity distribution and correlation function were performed with Dynamic Light Scattering (DLS) and were carried out with a Zetasizer NanoZS (Malvern Panalytical, Ltd, UK). Size measurements were conducted at a 173-degree scattering angle, with the appropriate settings for the solvent. Measurements were conducted at least 3 times with 30 s to 240 s of equilibration time. Samples were diluted 10-fold for measurements into ap 0.02 pm filtered water. Measurements were collected in Malvern Panalytical Inc 40pL Cuvettes (Fisher Scientific, NC0628994).

[0050] Rapamycin-loaded nanoparticles: Polymeric NPs were synthesized via FNP with a CIJ. To obtain NPs of 110 ± 20 nm loaded with Rapamycin, a concentration ratio of 3:4 RapamycimSynperonic PE / P48 was used in the solvent stream. To determine the encapsulation of the drug, an aliquot of the NPs was separated after dialysis and dried completely in a Speedvac25LEGAL02 / 47570581V1Attorney Docket No. 035052 / 640791SVC100 (Savant, US). The dried NPs were resuspended in acetonitrile to completely dissolve the NPs, then the Rapamycin content was determined by high performance liquid chromatography coupled with a diode array detector (HPLC-DAD) Agilent 1200 (Agilent Technology, US) at 277 nm. A calibration curve of increasing concentrations of Rapamycin standard was employed for the quantification.Example 2: Nanoparticle Uptake Assay

[0051] Nanoparticle uptake by endothelial cells: Healthy HUVECs and HUVECs expressing PIK3CA activating mutation were seeded at 200,000 cells per well in a 6 well sterile cell culture plate coated with 50 pg / mL of rat-tail derived type I collagen (Corning 354326) in 0.02 M acetic acid solution. Cells were cultured in EGM2 media incubated at 37 °C for two days prior to the application of orbital flow. Fresh EGM-2 (2 mL) was added into each well prior to the application of orbital flow. Orbital flow was applied by culturing ECs on an orbital shaker set to 200 rpm, which corresponds to a maximum wall shear stress of 15 Dynes / cm2. After 3 days of orbital flow, cells were treated with fluorescent Dil-loaded NPs diluted in fresh EGM-2 media. Cells were incubated at 37 °C for 8 hours. At the end of the incubation period, cells were rinsed once with PBS and were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) in PBS for 15 min at room temperature. For endpoint immunostaining, fixed cells were permeabilized and blocked with 0.1% Triton X-100 in 2% (w / v) BSA in PBS for 30 minutes at room temperature. Cells were incubated in primary antibodies against VE-cadherin (1 :500, v / v, F-8, Santa Cruz Biotechnology; 1 :250, diluted in 2% BSA blocking solution) for 2 hours at room temperature. Cells were subsequently rinsed three times over 15 min with PBS and were incubated in secondary antibodies (1 : 1000, v / v, goat anti-mouse immunoglobulin G conjugated to Alexa Fluor 647, Thermo Fisher Scientific) and DAPI (1 :1000, v / v, Thermo Fisher Scientific) diluted in blocking solution for 30 minutes at room temperature. Labeled cells and devices were rinsed three times over 15 min with PBS and were stored in 0.01% Sodium Azide solution diluted in PBS until imaging. Confocal z-stacks images of fluorescently labeled cells and nanoparticles (captured from top to bottom of endothelial cells) were acquired with an Olympus FV3000 laser scanning confocal microscope with a 20x UC Plan Fluor NA 0.7 air objective. Acquisition settings (laser power and gain of detector) were kept the same for each sample. A custom Imaris workflow used to quantify total Dil fluorescence intensity within each cell volume, defined by pixels within a volume26LEGAL02 / 47570581V1Attorney Docket No. 035052 / 640791 confined to the VE-cadherin positive boundary. Total fluorescence intensities of Dil within each segmented cells were measured using the total intensity quantification within Imaris.

[0052] Endocytosis inhibition: To inhibit internalization pathways, healthy HUVECs and HUVECs expressing PIK3CA activation mutations were incubated with endocytosis inhbitors. Cells were treated with 1 pM 5-(N-ethyl-N-isopropyl)-amiloride (EIP A), 10 pM PitStop2, 60 pM Dynasore, or dimethyl sulfoxide (DMSO) load controls in EGM2 during dosing of Dil-loaded nanoparticles. After 8 hrs, cells were rinsed once with PBS and were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. Cells were then stained, imaged, and quantified following the same procedure as the nanoparticle uptake assay.

[0053] MTT assay for cell viability: PIK3CAE545KHUVEC were seeded in 96 well plates at 3200 cells / well. After 24 hrs, 110 nm nanoparticles with encapsulated rapamycin were added to each well to result in concentrations of 1, 5, 10, 20, 50, 100, and 1000 nM rapamycin in EGM2. Cells were incubated with nanoparticles or free rapamycin in EGM2 at concentrations of 20, 50, and 1000 nM for 24 hrs. Cells were then washed with PBS prior to performing the MTT assay following manufacturer protocols (CyQuant MTT cell viability assay, Thermo Fisher), and absorbance was read at 570 nm using a plate reader. The cell viability percentage was calculated for each technical replicate as the raw absorbance minus the negative control (no cells in well) normalized by the positive control (no treatment) minus the negative control multiplied by 100.

[0054] Designation of a range of values includes all integers within or defining the range, and all subranges defined by integers within the range.

[0055] Unless otherwise apparent from the context, the term “about” encompasses values within a standard margin of error of measurement (e.g., SEM) of a stated value or variations ± 0.5%, 1%, 5%, or 10% from a specified value.

[0056] Compositions or methods “comprising” or “including” one or more recited elements may include other elements not specifically recited. For example, a composition that “comprises” or “includes” a protein may contain the protein alone or in combination with other ingredients.

[0057] The singular forms of the articles “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “an antigen” or “at least one antigen” can include a plurality of antigens, including mixtures thereof.

[0058] Statistically significant means p <0.05.27LEGAL02 / 47570581V1Attorney Docket No. 035052 / 640791

[0059] Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which the inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.28LEGAL02 / 47570581V1

Claims

Attorney Docket No. 035052 / 640791WHAT IS CLAIMED IS:

1. A method of selectively targeting a cell expressing upregulated PI3K activity in a subject in vivo, the method comprising: administering to the subject a plurality of nanoparticles having an average diameter from about 50 nm to about 220 nm, the nanoparticles comprising: one or more polymer layers; and, one or more pharmaceutical agents.

2. The method of claim 1, wherein the cell expressing upregulated PI3K activity is a mutated endothelial cell, a tumor associated endothelial cell, or a tumor cell.

3. The method of claim 2, wherein the cell expressing upregulated PI3K activity is an endothelial cell with PIK3CA mutation or TEK mutation.

4. The method of claim 2, wherein the mutated endothelial cell expresses elevated cell proliferation, elevated RAC1 activity or sprouting angiogenesis activity.

5. The method of claim 4, wherein the cell is present in a vascular lesion.

6. The method of claim 5, wherein the vascular lesion is caused by vascular malformations, a condition that results in malformation in vascular or lymphatic tissue or both.

7. The method of claim 6, wherein the malformation is present in one or more of a vein, an artery, a capillary, or a lymphatic vessel.

8. The method of any one of claims 1 to 7, wherein the nanoparticle is a polymeric nanoparticle further comprising a core, wherein the core comprises the one or more pharmaceutical agents, and the one or more polymer layers surround the core.

9. The method of claim 8, wherein one, two or three polymer layers surround the core.29LEGAL02 / 47570581V1Attorney Docket No. 035052 / 64079110. The method of claim 8, wherein the one or more polymer layers surrounding the core comprises polyethylene glycol (PEG).

11. The method of claim 8, wherein the agent is a precipitated hydrophobic drug, and the one or more polymer layers surrounding the core comprises a amphiphilic diblock co-polymer.

12. The method of claim 11, wherein the amphiphilic diblock co-polymer comprises a hydrophobic block, wherein the hydrophobic block comprises polylactide.

13. The method of claim 8, wherein the agent is a precipitated hydrophobic drug, and the one or more polymer layers surrounding the core comprises an amphiphilic triblock co-polymer.

14. The method of claim 13, wherein the amphiphilic triblock co-polymer comprises a hydrophobic block, wherein the hydrophobic block comprises polypropylene oxide.

15. The method of claim 13, wherein the amphiphilic triblock co-polymer comprises a hydrophobic block, wherein the hydrophobic block comprises polyethylene oxide.

16. The method of claim 8, wherein the nanoparticle comprises two pharmaceutical agents, wherein the agents are dual encapsulated active agents or are separate.

17. The method of claim 1, wherein the nanoparticle further comprises a core comprising the one or more pharmaceutical agents and poly-acrylic acid complexed with spermine, triethylamine, or Ca2+, or combinations thereof; and wherein the core is coated with the one or more polymer layers, wherein the polymer layers are each a PEG layer.

18. The method of claim 1, wherein the pharmaceutical agent is a PIK3CA inhibitor, mTORCl inhibitor, mT0RC2 inhibitor, mTORCl / 2 inhibitor, MEK inhibitors, or CDK4 / 6 inhibitor, or a combination of any two of the pharmaceutical agents.30LEGAL02 / 47570581V1Attorney Docket No. 035052 / 64079119. The method of claim 18, wherein the pharmaceutical agent is selected from the group consisting of Rapamycin, Alpelisib, Trametinib, Binimetinib, Selumetinib, Dabrafenib, Cobimetinib, Torkinib and Palbociclib, or a combination thereof.

20. The method of claim 19, wherein the pharmaceutical agent is Rapamycin or Alpelisib, or a combination of Rapamycin and Alpelisib.

21. The method of claim 20, wherein the pharmaceutical agent is Rapamycin.

22. The method of claim 1, wherein the plurality of nanoparticles have an average diameter of from about 60 nm to about 210 nm.

23. The method of claim 1, wherein the plurality of nanoparticles have an average diameter of about 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 or 210 nm.

24. The method of any one of claims 1, 22 or 23, wherein the plurality of nanoparticles has a PDI of from about 0.150 to about 0.240.

25. The method of any one of claims 1-24, wherein nanoparticles of the plurality are preferentially uptaken by the cell with upregulated PI3K activity.

26. The method of claim 1, wherein the nanoparticle further comprises: a core comprising Rapamycin or Alpelisib or a combination thereof, and poly-acrylic acid complexed with spermine, triethylamine, or Ca2+, or combinations thereof; wherein the core is coated with the one or more polymer layers, wherein the polymer layers are each a PEG layer; and the plurality of nanoparticles have an average diameter of between about 70 nm and about 120 nm.

27. The method of any one of claims 1-26, wherein the nanoparticles of the plurality are prepared by Flash Nanoprecipitation (FNP) and / or inverse Flash Nanoprecipitation (iFNP).31LEGAL02 / 47570581V1Attorney Docket No. 035052 / 64079128. A method of treating a disease associated with upregulated PT3K activity in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical agent in a nanoparticle formulation, wherein the nanoparticle formulation comprises a plurality of nanoparticles having an average diameter of from about 50 nm to about 220 nm, and wherein the nanoparticles comprise a polymer and one or more pharmaceutical agents.

29. The method of claim 28, wherein the nanoparticle formulation further comprises a pharmaceutically acceptable excipient.

30. The method of claim 28, wherein the plurality of nanoparticles have an average diameter of from about 60 nm to about 210 nm.

31. The method of claim 28, wherein the plurality of nanoparticles have an average diameter of about 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 or 210 nm.

32. The method of claim 31, wherein the plurality of nanoparticles has a PDI of from about 0.150 to about 0.240.

33. The method of claim 28, wherein the disease is selected from the group consisting of cancer, vascular malformations and lymphatic malformations.

34. The method of claim 33, wherein the disease is selected from the group consisting of vascular malformations and lymphatic malformations.

35. The method of claim 28, wherein the upregulated PI3K activity is a result of PIK3CA, TEK, KRAS, NRAS, or BRAF activating mutations in endothelial cells.

36. The method of any one of claims 28-35, wherein the nanoparticle is a polymeric nanoparticle further comprising a core, wherein the core comprises the one or more pharmaceutical agents, and wherein the one or more polymer layers surround the core.32LEGAL02 / 47570581V1Attorney Docket No. 035052 / 64079137. The method of claim 36, wherein one, two or three polymer layers surround the core.

38. The methods of claim 37, wherein the one or more polymer layers surrounding the core comprises polyethylene glycol (PEG).

39. The method of claim 28, wherein the nanoparticle comprises two pharmaceutical agents, wherein the agents are dual encapsulated active agents or are separate.

40. The method of claim 28, wherein the nanoparticle further comprises a core comprising the one or more pharmaceutical agents and poly-acrylic acid complexed with spermine, triethylamine, or Ca2+, or combinations thereof; and wherein the core is coated with the one or more polymer layers, wherein the polymer layers are each a PEG layer.

41. The method of claim 28, wherein the pharmaceutical agent is a PIK3CA inhibitor, mTORCl inhibitor, mTORC2 inhibitor, mTORCl / 2 inhibitor, MEK inhibitors, or CDK4 / 6 inhibitor, or a combination of any two of the pharmaceutical agents.

42. The method of claim 41, wherein the pharmaceutical agent is selected from the group consisting of Rapamycin, Alpelisib, Trametinib, Binimetinib, Selumetinib, Dabrafenib, Cobimetinib, Torkinib and Palbociclib, or a combination thereof.

43. The method of claim 42, wherein the pharmaceutical agent is Rapamycin or Alpelisib, or a combination of Rapamycin and Alpelisib.

44. The method of claim 43, wherein the pharmaceutical agent is Rapamycin.

45. The method of claim 28, wherein nanoparticles of the plurality are preferentially taken up by cells expressing the upregulated PI3K activity.

46. The method of claim 28, wherein the nanoparticle further comprises: a core comprising Rapamycin or Alpelisib or a combination thereof, and poly-acrylic acid complexed with spermine, triethylamine, or Ca2+, or combinations thereof;33LEGAL02 / 47570581V1Attorney Docket No. 035052 / 640791 wherein the core is coated with the one or more polymer layers, wherein the polymer layers are each a PEG layer; and the plurality of nanoparticles have an average diameter of between about 70 nm and about 120 nm.

47. The method of any one of claims 28-46, wherein the nanoparticles of the plurality are prepared by Flash Nanoprecipitation (FNP) and / or inverse Flash Nanoprecipitation (iFNP).34LEGAL02 / 47570581V1

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