Drug-lipid conjugated layer-by-layer nanoparticle for glioblastoma treatment

Abstract

Particles are provided that include a liposome having a negatively charged outer surface and a lipid-drug conjugate, a first layer of cationic polymer such as poly-L-arginine (PLR), that is non-covalently associated with the negatively charged outer surface of the liposome, and a second layer having a mixture of an anionic polymer and polyethylene glycol modified anionic polymer that is non-covalently associated with the first layer. The particles can be formulated as pharmaceutical compositions that are useful in methods that target neurological disorders such as brain tumors and other neurological diseases, and that can deliver and / or transport therapeutic drugs across the blood brain barrier.

Description

[0001] Drug-Lipid Conjugated Layer-by-Layer Nanoparticle for Glioblastoma Treatment

[0002] Statement of Government Support

[0003] This invention was made with government support under CA14051 awarded by the National Institutes of Health, and the government has certain rights in the invention. Additional funding support was provided by the University of Edinburgh (Grant C42454 / A28596 Ref. 7054685) and by the Natural Sciences and Engineering Research Council of Canada - AJP (Grant CGSD3-557538-2021).

[0004] Background

[0005] Neurological disorders such as brain tumors and neurological diseases are the leading global cause of years of life lost and second leading cause of death, according to data published in 2019. Development of new treatments for neurologic disorders has been challenging, largely due to poor drug transport across the blood-brain barrier (BBB) - the specialized vascular lining of the central nervous system. At least 95% of newly discovered candidate therapies are excluded from entering the brain - even in glioma tumors and neurological diseases commonly associated with “leaky” BBBs - and there is a critical need to develop drug carriers that can deliver therapeutics across the BBB.

[0006] Summary

[0007] In a first aspect, the disclosure provides a particle, comprising:

[0008] (a) a liposomal lipid bilayer comprising a negatively charged outer surface and a lipid-drug conjugate, wherein the lipid-drug conjugate comprises a covalent bond between the lipid and the drug;

[0009] (b) a first layer comprising a cationic polymer, wherein the cationic polymer is non-covalently associated with the negatively charged outer surface of the liposome; and

[0010] (c) a second layer, comprising a combination of an anionic polymer and a polyethylene glycol modified anionic polymer, wherein the second layer is non-covalently associated with the first layer.

[0011] In embodiments of the first aspect, the particle comprises a ratio of about 0.4 to about 0.8 weight equivalent of cationic polymer to liposome lipid bilayer. In some embodiments, the ratio comprises about 0.6 weight equivalent of cationic polymer to liposome lipid bilayer. In accordance with some further embodiments, the ratio of cationic polymer to liposome lipid bilayer is sufficient to yield a zeta potential greater than 30 mV. In some embodiments of the first aspect, the particle comprises a ratio of about 0.5 to about 1.5 weight equivalents of combined anionic polymer and polyethylene glycol modified anionic polymer to liposome lipid bilayer. In some embodiments, the ratio comprises about 0.8 to about 1.2 weight equivalent of combined anionic polymer and polyethylene glycol modified anionic polymer to liposome lipid bilayer. In some further embodiments, the ratio comprises about 1.0 weight equivalent of combined anionic polymer and polyethylene glycol modified anionic polymer to liposome lipid bilayer. In accordance with yet some further embodiments, the ratio of combined anionic polymer and polyethylene glycol modified anionic polymer to liposome lipid bilayer is sufficient to yield a zeta potential less than -30 mV.

[0012] In some embodiments of the first aspect, the second layer combination of anionic polymer and polyethylene glycol modified anionic polymer comprises a ratio of about 70:30 to about 40:60 weight equivalents of anionic polymer to polyethylene glycol modified anionic polymer. In some embodiments, the ratio comprises about 60:40 to about 50:50 weight equivalents of anionic polymer to polyethylene glycol modified anionic polymer. In yet further embodiments, the ratio comprises about 60:40 weight equivalents of anionic polymer to polyethylene glycol modified anionic polymer.

[0013] In some embodiments of the first aspect, the lipid comprising the lipid-drug conjugate comprises DPPT (l,2-dipalymitoyl-sn-glycero-3 -phosphothioethanol); cholesterol; DSPC ( 1 ,2-distearoyl-sn-glycero-3 -phosphocholine); DSPE ( 1 ,2-Distearoyl-sn-glycero-3 - phosphoethanolamine); DPPC (l,2-dipalmitoyl-glycero-3-phosphocholine); 18:0 propargyl PC (l,2-distearoyl-sn-glycero-3 -phosphocholine (N-propynyl)); 16:0 azidocaproyl PE (1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(6-azidohexanoyl)); 18:0 azidoethyl PC (l,2-distearoyl-sn-glycero-3 -phosphocholine (N-azidoethyl)); 16:0 DBCO PE (1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-dibenzocyclooctyl); DMPC (1,2- dimyristoyl-sn-glycero-3-phosphochline); POPC (1 -palmitoyl -2 -oleoyl-glycero-3 - phosphocholine); or DSPG (l,2-Distearoyl-sn-glycero-3-phosphoglycerol), or derivatives thereof, or any combination of one or more thereof. In some further embodiments, the lipid- drug conjugate comprises DPPT. In yet further embodiments the lipid-drug conjugate comprises DPPT and at least one additional lipid.

[0014] In a second aspect, the disclosure provides a particle, comprising:

[0015] (a) a liposomal lipid bilayer comprising l,2-dipalymitoyl-sn-glycero-3- phosphothioethanol (DPPT) conjugated covalently to a drug (i.e., a DPPT-drug conjugate); (b) a first layer comprising poly-L-arginine (PLR), wherein the PLR is non- covalently associated with the negatively charged outer surface of the liposome; and

[0016] (c) a second layer, comprising a combination of poly-L-glutamic acid (PLE) and poly-L-glutamate-b-polyethylene glycol (PLE-PEG), wherein the second layer is non- covalently associated with the first layer.

[0017] In some embodiments of the second aspect, the particle comprises a ratio of about 0.4 to about 0.8 weight equivalent of PLR to liposome lipid bilayer. In some embodiments of the second aspect, the particle comprises a ratio of about 0.6 weight equivalent of PLR to liposome lipid bilayer. In accordance with some further embodiments of the second aspect, the ratio of PLR to liposome lipid bilayer is sufficient to yield a zeta potential greater than 30 mV.

[0018] In some embodiments of the second aspect, the particle comprises a ratio of about 0.5 to about 1.5 weight equivalents of combined PLE and PLE-PEG to liposome lipid bilayer. In some embodiments of the second aspect, the particle comprises a ratio of about 0.8 to about 1.2 weight equivalent of combined PLE and PLE-PEG to liposome lipid bilayer. In some embodiments of the second aspect, the particle comprises or a ratio of about 1.0 weight equivalent of combined PLE and PLE-PEG to liposome lipid bilayer. In accordance with yet some further embodiments of the second aspect, the ratio of combined PLE and PLE-PEG to liposome lipid bilayer is sufficient to yield a zeta potential less than -30 mV.

[0019] In some embodiments of the second aspect, the PLE-PEG comprises a ratio of about 70:30 to about 40:60 weight equivalents of PLE:PEG. In some embodiments of the second aspect, the PLE-PEG comprises a ratio of about 60:40 to about 50:50 weight equivalents of PLE:PEG. In yet some further embodiments of the second aspect, the PLE-PEG comprises a ratio of about 60:40 weight equivalents of PLE:PEG.

[0020] In some embodiments of the above aspects and embodiments, the liposomal lipid bilayer comprises DPPT (l,2-dipalymitoyl-sn-glycero-3-phosphothioethanol); cholesterol; DSPC (l,2-distearoyl-sn-glycero-3 -phosphocholine); DSPE (l,2-Distearoyl-sn-glycero-3- phosphoethanolamine); DPPC (l,2-dipalmitoyl-glycero-3-phosphocholine); 18:0 propargyl PC (l,2-distearoyl-sn-glycero-3 -phosphocholine (N-propynyl)); 16:0 azidocaproyl PE (1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(6-azidohexanoyl)); 18:0 azidoethyl PC (l,2-distearoyl-sn-glycero-3 -phosphocholine (N-azidoethyl)); 16:0 DBCO PE (1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-dibenzocyclooctyl); DMPC (1,2- dimyristoyl-sn-glycero-3-phosphochline); POPC (l-palmitoyl-2-oleoyl-glycero-3- phosphocholine); or DSPG (l,2-Distearoyl-sn-glycero-3-phosphoglycerol), or derivatives thereof, or any combination of one or more thereof.

[0021] In some embodiments of the above aspects and embodiments, the drug comprises one or more of cytotoxins (including but not limited to dolastatins (including but not limited to dolastatin 10), auristatins (including but not limited to monomethyl auristatin E (MMAE), and monomethyl auristatin F (MMAF)), maytansines (including but not limited to ansamitocin, mertansine, and ravtansine), calicheamicins, and toxoids), angiogenesis inhibitors (including but not limited to axitinib, bevacizumab, cabozantinib, everolimus, lenalidomide, lenvatinib mesylate, pazopanib, ramucirumab, regorafenib, sorafenib, sunitinib, thalidomide, vandetanib, and ziv-aflibercept), anthracyclines (including but not limited to daunorubicin, doxorubicin, and epirubicin), immune checkpoint inhibitors (including, but not limited to, pembrolizumab, nivolumab, and cemiplimab as anti-PD-1 antibodies, ipilimumab as an anti-CTLA-4 antibody, and atezolizumab, avelumab, and durvalumab as anti-PD-Ll antibodies), microtubule stabilizers (including by not limited to docetaxel or paclitaxel), and other cancer growth inhibitors including but not limited to tyrosine kinase inhibitors (including but not limited to alectinib, brigatinib, ceritinib, crizotinib, entrectinib, lorlatinib, ALK, I, bosutinib, dasatinib, imatinib, nilotinib, ponatinib, afatinib, dacomitinib, erlotinib, gefitinib, lapatinib, neratinib, osimertinib, vandetanib, gilteritinib, midostaurin, erdafitinib, ruxolitinib, larotrectinib, axitinib, carbozantinib, lenvatinib, pazopanib, regorafenib, sorafenib, sunitinib, dabrafenib, encorafenib, vemurafenib, acalabrutinib, ibrutinib, binimetinib, cobimetinib, trametinib, abemaciclib, palbociclib, ribociclib, or eCF506), proteasome inhibitors (including but not limited to ortezomib, carfizomib, ixazomib, delanzomib, oprozomib, and marizomib), mTOR inhibitors (including but not limited to everolimus, sirolimus, temsirolimus, everolimus, sirolimus, sirolimus protein-bound, and everolimus), PI3K inhibitors (including but not limited to copanlisib, alpelisib, idelalisib, duvelisib and umbralisib), histone deacetylase inhibitors (including but not limited to vorinostat, romidepsin, panobinostat, and belinostat), and Hedgehog pathway blockers (including but not limited to vismodegib, sonidegib, and glasdegib), derivatives thereof, combinations thereof, and / or pharmaceutically acceptable salts thereof. In some further embodiments, the drug comprises a cytotoxin. In yet some further embodiments, the drug comprises a cancer growth inhibitor. In some further embodiments, the drug comprises an angiogenesis inhibitor. In some further embodiments, the drug comprises an auristatin. In yet some further embodiments, the drug comprises maleimidocaproyl-monomethyl auristatin F (mc-MMAF). In some embodiments of the above aspects and embodiments, the particle comprises up to about 1.0 mol % lipid-drug conjugate in the liposome lipid bilayer. In some further embodiments the lipid-drug conjugate comprises DPPT-MMAF.

[0022] In embodiments of the above aspects and embodiments, the particle has a diameter range between about 50 nm to about 500 nm. In some further embodiments, the particle has a diameter range between about 80 to about 120 nm. In yet some further embodiments, the particle has a diameter range between about 90 to about 100 nm.

[0023] In a third aspect, the disclosure provides a pharmaceutical composition, comprising (a) a plurality of the particles in accordance with any of the aspects and embodiments of the disclosure; and (b) a pharmaceutically acceptable carrier.

[0024] In some embodiments, the pharmaceutical composition is formulated to avoid and / or reduce first pass metabolism of the drug.

[0025] In some embodiments, the pharmaceutical composition is formulated for intravenous administration.

[0026] In some further embodiments, the pharmaceutical composition is formulated for convection-enhanced delivery (CED).

[0027] In a fourth aspect, the disclosure provides a method for treating a cancer in the brain, comprising administering to a subject having a cancer in the brain the particle or pharmaceutical composition in accordance with any of the aspects and embodiments of the disclosure in an amount effective to treat the cancer. In some further embodiments, the cancer in the brain is a glioma or a glioblastoma. In yet some further embodiments, the particle or pharmaceutical composition is administered to the subject intravenously. In some other embodiments, the particle or pharmaceutical composition is administered to the subject by convection-enhanced delivery (CED). In some embodiments, the subject is a human.

[0028] The disclosure also provides methods for making the particle in accordance with any of the aspects and embodiments of the disclosure, comprising covalently attaching the drug to the lipid to generate the lipid-drug conjugate, and incorporating the lipid-drug conjugate into the liposome. In some further embodiments, the method can further comprise loading an additional therapeutic into the particle.

[0029] Other aspects and embodiments will be apparent to one of skill in the art in view of the detailed description and illustrative examples that follow.

[0030] Brief Description of the Figures Figure 1 depicts a schematic of an illustrative nanoparticle lipid-drug conjugate, DPPT-MMAF, that is incorporated into an anionic liposome (e.g., ~ 90 nm), further functionalized with an LbL structure comprising polyelectrolytes that prolong brain retention and improve the spatial and cellular distribution in glioblastoma brain tumors.

[0031] Figure 2. Synthesis of DPPT-MMAF. (A) Depicts a reaction scheme showing the organic phase conjugation reaction between the thiol-containing DPPT and maleimide- functionalized mc-MMAF to form an example lipid-drug conjugate, DPPT-MMAF. (B) The 1 : 1 stoichiometry of the reaction was confirmed by monitoring the consumption of mc- MMAF following reaction with specified molar equivalents of DPPT.

[0032] Figure 3. Formulation of MMAF -conjugated LbL NPs. (A) Schematic showing three different types of liposomes that are described and illustrated herein. Stars indicate MMAF. (B) Shows the Z-average diameter and polydispersity index (PDI) for the three liposome formulations measured by dynamic light scattering. (C) Shows Zeta potential for the three liposome formulations as measured by dynamic light scattering. (D) Shows relative thiol content of the three different liposome formulations as measured by an ABD-F thiol detection assay. (E) Provides a schematic illustrating one approach to layer-by-layer functionalization of MMAF-conjugated liposomes. (F) illustrates the Z-average diameter and polydispersity index of the NPs following each step of LbL assembly. (G) illustrates zeta potential of NPs following each step of the LbL assembly. (H) Illustrates the Z-average diameter of the exemplary MMAF-conjugated liposomes and LbL functionalized MMAF-liposomes with outer layers 60% (w / w) PLE and 40% (w / w) PLE-PEG (referred to as co-PLE NPs) following overnight incubation in artificial cerebrospinal fluid at 37 °C.

[0033] Figure 4. In vitro cytotoxicity of MMAF formulations. (A) Cell viability of MMAF formulations against (i-iv) glioblastoma cells and (v-i) healthy neural cells. Viability was quantified through metabolic activity measured with a resazurin assay. (B) Comparison of IC50s of free MMAF, EGFR-MMAF, liposome-MMAF, and LbL-MMAF in each cell line. LbL-MMAF NPs demonstrated striking potency against all cancer cell lines regardless of EGFR mutation status, with reduced sensitivity against healthy cells.

[0034] Figure 5. In vivo efficacy of LbL-MMAF NPs. (A) H&E staining of a GBM12 intracranial tumor 7-days post inoculation. (B) Experimental timeline for GBM12 efficacy study. CED was performed on day 8 using dextrose, free MMAF, liposome-MMAF, LbL- MMAF, and EGFR-MMAF. (C) T2 -weighted MRI scans of mice at various time points after tumor inoculation and (D) the 3D tumor volume of tumors detected on MRI confirms the delayed tumor growth. (E) Body weight of mice following tumor inoculation and CED for the duration of the study. (F) Kaplan-Meier survival curve shows prolonged survival following CED of LbL-MMAF NPs when compared to control groups.

[0035] Figure 6. Distribution and activity of LbL-MMAF NPs in GBM12 glioblastoma tumors. (A) Cryofluorescent microscopy staining of a GBM12 tumor-bearing mouse brain section 24 hours following CED with LbL-MMAF NPs. Nuclei were stained with DAPI, tumor cells with anti-human vimentin, and MMAF with a rabbit anti-MMAF antibody. Nanoparticles were visualized via incorporation of a fluorescent lipid, PE-BDP650 / 665 at 0.2% (mol / mol) in the liposome core. (B) Cryofluorescent immunostaining of cleaved- caspase 3 confirms cytotoxicity of LbL NPs within GBM12 tumors, while dextrose injection shows minimal staining. (C) Neuron staining with NeuN 10 days following CED of LbL NPs shows no significant neuron loss throughout the infused striatum.

[0036] Figure 7. MMAF distribution, activity, and retention impact anti-GBM efficacy in vivo. A) Widefield fluorescence micrographs of brain sections (10 pm thickness) 24 hours post-CED, showing the distribution of the carrier (NP or ADC) and MMAF in GBM12- bearing mice treated with dextrose, Lipo-MMAF, LbL-MMAF, or EGFR-MMAF. Sections are labeled for nuclei (DAPI), anti-human vimentin (hVim), nanoparticle fluorescence or anti-human IgG (carrier), and anti-MMAF (MMAF). LbL-MMAF shows more uniform tumor penetration compared to Lipo-MMAF, which remains confined to the tumor core, while EGFR-MMAF spreads throughout the hemisphere. B) High magnification fluorescent micrographs of tumors 24-hours post-CED stained for cleaved caspase-3 (cCasp-3), antihuman vimentin, and nanoparticle fluorescence or anti-human IgG (carrier), showing significantly increased apoptotic activity with LbL-MMAF compared to both Lipo-MMAF and EGFR-MMAF. C) Quantification of the percentage of cCasp-3+ apoptotic cells in the tumor region shows significantly higher apoptosis in LbL-MMAF-treated mice compared to Lipo-MMAF and EGFR-MMAF. D) Quantification of the percentage of apoptotic cells that are hVim+, indicating higher tumor specificity of LbL-MMAF and EGFR-MMAF compared to Lipo-MMAF. Error bars in panels C and D represent the standard deviation from the arithmetic mean of four biological replicates (*p < 0.05, one-way ANOVA with Welch’s correction). E) Fluorescence micrographs showing the retention of LbL NPs in tumor tissue at 5 and 10 days post-CED. LbL NP signal remains colocalized with MMAF at both time points, though fluorescence intensity decreases by day 10. F) Comparative images of EGFR- MMAF-treated mice show the initial distribution of MMAF and ADC at day 5, which diminishes by day 10, leaving a residual population of tumor cells. Nuclei (DAPI) and hVim are labeled in all panels. Detailed Description

[0037] As used herein and unless otherwise indicated, the terms “a” and “an” are taken to mean “one”, “at least one” or “one or more”. Unless otherwise required by context, singular terms used herein shall include pluralities and plural terms shall include the singular.

[0038] Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is, in the sense of “including, but not limited to”. Additionally, the words “herein,” “above” and "below" and words of similar import, when used in this application, shall refer to this application as a whole and not to any particular portions of this application.

[0039] All aspects and embodiments of the disclosure can be used in various combination, unless the context clearly dictates otherwise.

[0040] As used herein, "about" will refer to an approximate value that allows for some amount of variation, but not so much variation as to have a material effect on the associated function or property. Thus, in some embodiments, a value or range of “about” a recited value or range can allow for up to 10% (plus or minus) the particular value or range, as long as that amount of variation does not have a material effect on the associated property. In some embodiments, “about” allows for 5% (plus or minus) the particular value or range, or in some embodiments, less than 5% of the particular value or range.

[0041] In a general sense, the disclosure provides particles, (e.g., nanoparticles) comprising a liposomal core that comprises a covalently bound lipid-drug conjugate, wherein the liposomal core is functionalized with two or more layers of polyelectrolyte layers having different charges. The layers of polyelectrolyte are applied in a layer-by-layer (UbU) deposition and comprise a first polycationic layer that is non-covalently associated with the liposomal core, and a second (or outer) polyanionic layer that is non-covalently associated with the first polycation layer and that comprises two different polyanionic polymers. The nanoparticles can be formulated as pharmaceutical compositions for delivery to the brain. As detailed by the illustrative examples below, the polyelectrolyte functionalization of the nanoparticle cores can provide for enhanced uptake, delivery, stability, and retention in target cells and, when combined with the lipid-drug conjugate, can provide for effective treatment of neurological disorders and brain tumors in particular.

[0042] In an aspect, the disclosure provides a particle, comprising: (a) a liposomal lipid bilayer comprising a negatively charged outer surface and a lipid-drug conjugate, wherein the lipid-drug conjugate comprises a covalent bond between the lipid and the drug;

[0043] (b) a first layer comprising a cationic polymer, wherein the cationic polymer is non-covalently associated with the negatively charged outer surface of the liposome; and

[0044] (c) a second layer, comprising a combination of an anionic polymer and a polyethylene glycol modified anionic polymer, wherein the second layer is non-covalently associated with the first layer.

[0045] As discussed herein, particles falling within the scope of the disclosure comprise a combination of a covalently conjugated lipid-drug conjugate incorporated as part of the liposomal lipid bilayer and differently charged polyelectrolyte layers, deposited layer-by- layer (typically referred to herein as, LbL NPs or LbL particles), which allows for substantially higher degree of uptake, targeting, and stabilizing functionalization than previously reported strategies. The targeting capacity, circulation, and stability benefits of the polymer layers are shown to significantly improve accumulation in target tissue (e.g., brain tumors) following administration in an animal model, and are shown to exert a significant therapeutic effect with an exemplary lipid-drug conjugate (e.g., DPPT-mcMMAF). Particles in accordance with the disclosure solve the problem of inefficient targeting and poor accumulation of therapies from the blood into the brain space - crossing the “blood-brain barrier” (BBB), which is a frequent reason that candidate therapeutics fail to progress through, for example, glioblastoma (GBM) treatment efficacy trials. By boosting transport across the BBB, as well as improving selective uptake by the cancer cells rather than healthy brain cells, these targeted particles enable translation of a wide variety of candidate drugs and can also reduce potential side effects (e.g., non-targeted cell toxicity).

[0046] Liposomes are spherical lipid vesicles approximately 50-500 nm in diameter particle size composed of one or more lipid bilayers, as a result of emulsifying natural or synthetic lipids in an aqueous medium. Liposomes have an aqueous core. Therapeutic drugs can be loaded into the aqueous core of the liposome or into the lipid bilayers, depending on the molecular properties of the therapeutic. Thus, in various embodiments, the liposome has a diameter of between about 50 nm and about 500 nm; or about 50 nm and about 250 nm; or about 50 nm and about 150 nm; or about 50 nm and about 100 nm. In other embodiments, the measured diameter of the particle (i.e., lipid-drug liposome conjugate-polycation-mixed polyanion) ranges between about 80 nm to about 500 nm, or from about 100 nm to about 150 nm. In some non-limiting embodiments, the liposome or particle can have a diameter of at least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, or about 525 nm.

[0047] In accordance with some of the aspects and embodiments described herein, the polycationic layer can comprise any polymer having a cationic charge under typical physiological conditions (e.g., physiological pH, ionic strength, etc.) and that can associate through electrostatic interaction with the outer surface of the lipid bilayer. Some non-limiting examples of polymers that can comprise the first polycationic layer include, chitosan, polyamidoamine (PAMAM) dendrimers, polyethylenimine (PEI), protamine, polyacrylamide, polyacrylamide copolymers, poly (arginine), poly(histidine), poly(lysine), poly(beta- aminoesters), poly(allylamine hydrochloride), poly(vinylamine), poly(2- (dimethylamino)ethyl methacrylate), poly(vinylamine), and other cationic celluloses and peptides, as well as derivatives thereof, and the like.

[0048] In some embodiments, the ratio of cationic polymer to liposome lipid bilayer (used interchangeably with “liposome core” herein) in the particles may be any ratio that yields a zeta potential greater than 30 mV as measured by dynamic light scattering (DLS). In some embodiments, the particle has a ratio of about 0. 5 to about 1 weight equivalent of cationic polymer to liposome lipid bilayer, yielding a measured increase in nanoparticle diameter between 1-50 nm. In other embodiments, the particle has a ratio of about 0.1 to about 0.8 (i.e., 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, or 0.8) weight equivalent of cationic polymer to liposome lipid bilayer, yielding a measured increase in particle diameter of about 5 to about 30 nm.

[0049] In some further embodiments, the first layer comprises poly-L-arginine hydrochloride (PLR) polymers having the structure:

[0050] . Poly-L-arginine hydrochloride (PLR).

[0051] In accordance with some of the aspects and embodiments described herein, the polyanionic layer can comprise a combination of a polyanionic polymer and modified polyanionic polymer, wherein the combination improves uptake and selectivity for target cells (e.g., glioblastoma cells) as well as colloidal stability under physiological conditions. In some non-limiting embodiments the polyanionic layer can comprise alginates, hyaluronates acid, heparin, heparin sulfate, chondroitin sulfate, dextran sulfate, sulfonated lignin, poly(meth)acrylates, oxidized celluloses, carboxymethyl cellulose, polyaspartate, polyglutamate, polyacrylates, alginates, polystyrenesulfonates, polysialic acid, carboxymethyl chitosan, polymannuronic acid, polyguluronic acid, fucoidan, as well as derivatives thereof, and the like.

[0052] In some further embodiments, the modified polyanionic polymer comprises a polyethylene glycol derivative.

[0053] In some yet further embodiments, the polyanionic layer comprises poly-L-glutamate (PLE) and poly-L-glutamate-b-polyethylene glycol (PLE-PEG).

[0054] In embodiments, the ratio of polyanionic layer to liposome lipid bilayer in the particles may be any ratio that yields a zeta potential less than -30 mV as measured by DLS. In some embodiments, the particle has a ratio of about 0.5 to about 1.5 weight equivalents (e.g., 0.5., 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.15, 1.2, 1.25, 1.3, 1.35, 1.4, 1.45, or 1.5) weight equivalent of combined anionic polymer and polyethylene glycol modified anionic polymer to liposome lipid bilayer. In some embodiments the particle has a ratio of about 0.8 to about 1.2 weight equivalent of combined anionic polymer and polyethylene glycol modified anionic polymer to liposome lipid bilayer, or a ratio of about 1.0 weight equivalent of combined anionic polymer and polyethylene glycol modified anionic polymer to liposome lipid bilayer that yields the zeta potential of less than -30 mV.

[0055] In some embodiments, the combined anionic polymer and polyethylene glycol modified anionic polymer comprises a ratio of about 70:30 to about 40:60 weight equivalents of anionic polymer to polyethylene glycol modified anionic polymer (e.g., 70:30, 65:35, 60:40, 55:45, 50:50, 45:55, or 40:60). In some embodiments, the combined anionic polymer and polyethylene glycol modified anionic polymer comprises a ratio of 60:40.

[0056] Any liposome having a negatively charged outer surface can be used in the particles as suitable for an intended use. In one embodiment, the liposomal lipid bilayer comprises DPPT (l,2-dipalymitoyl-sn-glycero-3-phosphothioethanol); cholesterol; DSPC (1,2- distearoyl-sn-glycero-3-phosphocholine); DSPE (l,2-Distearoyl-sn-glycero-3- phosphoethanolamine); DPPC (l,2-dipalmitoyl-glycero-3 -phosphocholine); DMPC (1,2- dimyristoyl-sn-glycero-3-phosphochline); POPC (1 -palmitoyl -2 -oleoyl-glycero-3 - phosphocholine); or DSPG (l,2-Distearoyl-sn-glycero-3-phosphoglycerol), or derivatives thereof, or any combination of one or more thereof, in amounts and / or ratios suitable for number average diameters between 50-100 nm as determined by DLS, and polydispersity index (PDI) less than 0.2. In one non-limiting embodiment, a suitable lipid bilayer formulation comprises DPPT and a lipid-drug conjugate. In some further embodiments the lipid-drug conjugate comprises DPPT.

[0057] In some further embodiments, the particles of the disclosure can further comprise any suitable BBB-targeting moiety such as, for example, a targeting peptide that can be covalently and / or electrostatically coupled to the outer layer and that provides a stable nanoparticle (zeta potential less than -30 mV). In accordance with such embodiments the particles can further comprise any targeting peptide known in the art to be effective in targeting invasive cells such as, for example, tumorigenic and / or cancerous cells. In such embodiments, the targeting peptides can be electrostatically attached to the particles by mixing the peptides (e.g., positively charged peptides) with the polyanionic outer layer of the particles, and allow the attraction of opposite charges to stably stick the two components together.

[0058] While the particles of embodiments of the disclosure relate to liposomes and nanoparticles that comprise a covalently bound lipid-drug conjugate as the active agent, some embodiments may further comprise one or more therapeutic drugs loaded into the aqueous core of the liposome or into the lipid bilayers, depending on the molecular properties of the therapeutic. In some embodiment, the additional therapeutic can be loaded into liposome aqueous core by adding the drug to the dried lipid fdm before rehydration in water. In other embodiments, the additional therapeutic can be loaded into the liposome lipid bilayer by adding it to the lipid film before liposome rehydration.

[0059] In some embodiments, the particles of the disclosure are particularly designed for administration to the brain, and particularly for treating brain tumors. Thus, in such embodiments, the lipid-drug conjugate comprises one or more chemotherapeutic. Any chemotherapeutic suitable for an intended treatment can be used, including but not limited to one or more therapeutic selected from the group consisting of angiogenesis inhibitors (including but not limited to axitinib, bevacizumab, cabozantinib, everolimus, lenalidomide, lenvatinib mesylate, pazopanib, ramucirumab, regorafenib, sorafenib, sunitinib, thalidomide, vandetanib, and ziv-aflibercept), anthracy clines (including but not limited to daunorubicin, doxorubicin, and epirubicin), immune checkpoint inhibitors (including, but not limited to, pembrolizumab, nivolumab, and cemiplimab as anti-PD-1 antibodies, ipilimumab as an anti- CTLA-4 antibody, and atezolizumab, avelumab, and durvalumab as anti-PD-Ll antibodies), microtubule stabilizers (including by not limited to docetaxel or paclitaxel), and other cancer growth inhibitors including but not limited to tyrosine kinase inhibitors (including but not limited to alectinib, brigatinib, ceritinib, crizotinib, entrectinib, lorlatinib, ALK, I, bosutinib, dasatinib, imatinib, nilotinib, ponatinib, afatinib, dacomitinib, erlotinib, gefitinib, lapatinib, neratinib, osimertinib, vandetanib, gilteritinib, midostaurin, erdafitinib, ruxolitinib, larotrectinib, axitinib, carbozantinib, lenvatinib, pazopanib, regorafenib, sorafenib, sunitinib, dabrafenib, encorafenib, vemurafenib, acalabrutinib, ibrutinib, binimetinib, cobimetinib, trametinib, abemaciclib, palbociclib, ribociclib, or eCF506), proteasome inhibitors (including but not limited to ortezomib, carfizomib, ixazomib, delanzomib, oprozomib, and marizomib), mTOR inhibitors (including but not limited to everolimus, sirolimus, temsirolimus, everolimus, sirolimus, sirolimus protein-bound, and everolimus), PI3K inhibitors (including but not limited to copanlisib, alpelisib, idelalisib, duvelisib and umbralisib), histone deacetylase inhibitors (including but not limited to vorinostat, romidepsin, panobinostat, and belinostat), and Hedgehog pathway blockers (including but not limited to vismodegib, sonidegib, and glasdegib), or pharmaceutically acceptable salts thereof.

[0060] As discussed herein, the benefit and therapeutic efficacy of the LbL nanoparticles can also rely on selecting a suitable drug as part of the lipid-drug conjugate. The illustrative embodiments described in the Examples demonstrates that cytotoxic agents that are typically cell-impermeable and have a risk of off-target toxicity (e.g., auristatins, and monomethyl auristatin F (MMAF) in particular) can be incorporated into the particles of the disclosure and effectively delivered to targeted cells. Thus, in some embodiments, the lipid-drug conjugate comprises an auristatin or a derivative thereof. In some further embodiments, the lipid-drug conjugate comprises monomethyl auristatin F (MMAF) or a derivative thereof. In yet further embodiments, the lipid-drug conjugate comprises maleimidocaproyl-monomethyl auristatin F (mc-MMAF). As shown in the examples that follow, DPPT-mcMMAF (or alternatively herein, “DPPT-MMAF”) was used to exemplify the particles of the disclosure. The example demonstrates that DPPT-mcMMAF can comprise an amount of the lipid bilayer of the particles of the disclosure, and its incorporation into such particles does not interfere either the integrity of the liposome, the targeting of the LbL-NPs, or with the cytostatic effect of MMAF on glioblastoma cells. As the data shows, the particles in accordance with that example embodiment and had effects that were improved relative to other controls (e.g., MMAF and other liposome / NP formulations).

[0061] The amount of the lipid-drug comprising the lipid bilayer can vary widely depending on the therapeutic drug and / or the lipid to which it is conjugated. In some non-limiting embodiments, the particle comprises up to about 3% (by weight or alternatively by mol) of the liposomal lipid bilayer, embodiments, the particle comprises between about 0.5 % to about 3 % lipid-drug conjugate of the liposome lipid bilayer, or between about 0.5 % to about 2.5 %, or between about 0.5 % and about 2.0 %, or between about 0.5 % and about 1.5 %., or between about 0.5 % and about 1.0 %. In some embodiments, the particle comprises about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%„ 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, or about 3% (by weight or alternatively by mol) of the liposomal lipid bilayer. In yet some further embodiments, the particle comprises 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5% (by weight or alternatively by mol) of the liposomal lipid bilayer.

[0062] In accordance with some example embodiments of this aspect, the disclosure provides a particle comprising: a) a liposomal lipid bilayer comprising l,2-dipalymitoyl-sn-glycero-3- phosphothioethanol (DPPT) conjugated covalently to a drug;

[0063] (b) a first layer comprising poly-L-arginine (PLR), wherein the PLR is non- covalently associated with the negatively charged outer surface of the liposome; and

[0064] (c) a second layer, comprising a combination of poly-L-glutamic acid (PLE) and poly-L-glutamate-b-polyethylene glycol (PLE-PEG), wherein the second layer is non- covalently associated with the first layer.

[0065] In yet further embodiments, the drug covalently conjugated to DPPT comprising mc- MMAF.

[0066] In another aspect, the disclosure provides pharmaceutical composition, comprising

[0067] (a) a plurality (i.e., at least 2, 5, 10, 50, 100, 1000, 5000, 10,000, 50,000, 100,000 or more) of the particles of any embodiment herein; and

[0068] (b) a pharmaceutically acceptable carrier.

[0069] In some further embodiments, the pharmaceutical composition and / or an amount of the plurality of the one or more particles can further comprise one or more additional therapeutics, or a pharmaceutically acceptable salts thereof, that is loaded within the core and / or the lipid bilayer of the liposomal lipid bilayer.

[0070] As used herein, the term “pharmaceutically acceptable carrier” refers to a pharmaceutically-acceptable material, composition or vehicle for administration of therapeutic described herein. Pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like which are compatible with the activity of the active agent and are physiologically acceptable to the subject. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (i) sugars, such as lactose, glucose and sucrose; (ii) starches, such as com starch and potato starch; (iii) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (iv) powdered tragacanth; (v) malt; (vi) gelatin; (vii) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (viii) excipients, such as cocoa butter and suppository waxes; (ix) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (x) glycols, such as propylene glycol; (xi) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (xii) esters, such as ethyl oleate and ethyl laurate; (xiii) agar; (xiv) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (xv) alginic acid; (xvi) pyrogen-free water; (xvii) isotonic saline; (xviii) Ringer's solution; (xix) ethyl alcohol; (xx) pH buffered solutions; (xxi) polyesters, polycarbonates and / or polyanhydrides; (xxii) bulking agents, such as polypeptides and amino acids (xxiii) semm component, such as semm albumin, HDL and LDL; (xxiv) C2-C12 alcohols, such as ethanol; and (xxv) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation.

[0071] Pharmaceutically acceptable carriers can vary in a formulation described herein, depending on the administration route. The formulations described herein can be delivered via any administration mode known to a skilled practitioner. For example, the formulations described herein can be delivered in a systemic manner, via administration routes such as, but not limited to, oral, and parenteral, including intravenous, intramuscular, intraperitoneal, intradermal, and subcutaneous.

[0072] In some embodiments, the pharmaceutical composition is formulated for an administration route that avoids and / or limits first pass metabolism. In some embodiments the pharmaceutical composition is formulated administration by insufflation, suppository, injection (intramuscular, intraperitoneal, intravenous, etc.), inhalation, intranasal, intraocular, transdermal, buccal, sublingual, and / or convection-enhanced delivery (CED).

[0073] In one embodiment, the pharmaceutical composition is formulated for administration by injection. In one embodiment, the pharmaceutical composition is formulated for administration by convection-enhanced delivery.

[0074] When administering parenterally, a formulation described herein can be generally formulated in a unit dosage injectable form (solution, suspension, emulsion). The formulations suitable for injection include sterile aqueous solutions or dispersions. The carrier can be a solvent or dispersing medium containing, for example, water, cell culture medium, buffers (e.g., phosphate buffered saline), polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof. In some embodiments, the pharmaceutical carrier can be a buffered solution (e.g., PBS). Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.

[0075] The formulations can also contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, preservatives, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as “REMINGTON'S PHARMACEUTICAL SCIENCE”, 17th edition, 1985, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation. With respect to formulations described herein, however, any vehicle, diluent, or additive used should have to be biocompatible with the active agents described herein. Those skilled in the art will recognize that the components of the formulations should be selected to be biocompatible with respect to the active agent. This will present no problem to those skilled in chemical and pharmaceutical principles, or problems can be readily avoided by reference to standard texts or by simple experiments (not involving undue experimentation).

[0076] For in vivo administration, the formulations described herein can be administered with a delivery device, e.g., a syringe. Accordingly, an additional aspect described herein provides for delivery devices comprising at least one chamber with an outlet, wherein at least one chamber comprises a pre-determined amount of any formulation described herein and the outlet provides an exit for the formulation enclosed inside the chamber. In some embodiments, a delivery device described herein can further comprise an actuator to control release of the formulation through the outlet. Such delivery device can be any device to facilitate the administration of any formulation described herein to a subject, e.g., a syringe, a dry powder injector, a nasal spray, a nebulizer, or an implant such as a microchip, e.g., for sustained-release or controlled release of any formulation described herein.

[0077] In another aspect, the disclosure provides methods for treating a cancer in the brain, comprising administering to a subject having a cancer in the particle or pharmaceutical composition of any embodiment herein, in an amount effective to treat the cancer. In one embodiment, the one or more lipid-drug conjugate comprises DPPT-mcMMAF. In another embodiment, the cancer in the brain is a glioma or a glioblastoma. In a further embodiment, the administration comprises administration by CED.

[0078] As used herein, "treat" or "treating" means accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting or preventing development of symptoms characteristic of the disorder(s) being treated; (c) inhibiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting or preventing recurrence of the disorder(s) in patients that have previously had the disorder(s); and (e) limiting or preventing recurrence of symptoms in patients that were previously symptomatic for the disorder(s). When the method comprises treating cancer, "treat" or "treating" means accomplishing one or more of the following: (a) reducing the size or volume of tumors and / or metastases in the subject; (b) limiting any increase in the size or volume of tumors and / or metastases in the subject; (c) increasing survival; (d) reducing the severity of symptoms associated with cancer; (e) limiting or preventing development of symptoms associated with cancer; and (f) inhibiting worsening of symptoms associated with cancer. The methods can be used to treat any suitable cancer, including but not limited to glioblastoma or glioma.

[0079] The subject may be any subject that has a relevant disorder. In one embodiment, the subject is a mammal, including but not limited to humans, dogs, cats, horses, cattle, etc. In one embodiment, the subject is a human subject.

[0080] The disclosure also provides methods for making the particles comprising the covalently attached lipid-drug conjugate of the disclosure. In some embodiments, the methods comprise any method that is effective for covalently binding a drug to a lipid. For example, a lipid comprising a reactive moiety (e.g., amide, thiol, carboxylate) or a linker, and an active agent that comprises or that can be derivatized to include a reactive group or a coupling agent can be selected and used to generate the conjugate, using known standard synthetic techniques (e.g., conjugation chemistry, antibody-drug conjugate chemistry, etc.; See, for example, Matsuda Y., and Mendelsohn B.A. “An overview of process development for antibody-drug conju-gates produced by chemical conjugation technology”. Exp. Opin. Biol. Ther. 2021;21:963-975; Adhikari, P., et al., “Nano lipid-drug conjugate: An integrated review. International Journal of Pharmaceutics,” 2017; 529(1-2), 200-217.; and Fujii, T., et al., “AJICAP Second Generation: Improved Chemical Site-Specific Conjugation Technology for Antibody-Drug Conjugate Production.” Bioconjuate Chem., 2023, 34: 728-738). Other types of common conjugation reactions include coupling of amines, thiols, esters, and hydroxides. Other strategies can introduce a unique functional group or linker onto a lipid, and couple via this unique functional group. In some embodiments, the drug and / or the lipid comprises a reactive group that can be coupled under appropriate reaction conditions.

[0081] Examples

[0082] Materials and Reagents l,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC), l,2-distearoyl-sn-glycero-3- phospho-(l'-rac -glycerol) (sodium salt) (DSPG), cholesterol, l,2-distearoyl-sn-glycero-3- phosphoethanolamine (DSPE), l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPT), and l-stearoyl-2-[(E)-4-(4-((4-butylphenyl)diazenyl)phenyl)butanoyl]-sn-glycero-3- phosphocholine (Azo-PC) were purchased from Avanti. BDP 650 / 665 DBCO and BDP Fl maleimide were purchased from Lumiprobe. Poly(L-arginine hydrochloride) (38 kDa), poly(L-glutamic acid sodium salt) (15 kDa), and methoxy-poly(ethylene glycol) -block- poly(L-glutamic acid sodium salt) (PLE 15 kDa-b-PEG 5 kDa) were purchased from Alamanda Polymers. Whatman Nucleopore™ polycarbonate hydrophilic membranes (400, 200, 100, and 50 nm sizes) were purchased from GE. Tangential flow fdtration fdters (D02- E100-05-N and C02-E100-05-N) were purchased from Repligen. DTS 1070 folded capillary zeta cells were purchased from Malvern. Monomethylauristatin F (MMAF) and Depatuxizumab mafodotin (EGFR-MMAF) were purchased from Selleck Chem. Maleimidocaproyl monomethylauristatin F (mcMMAF) was purchased from BroadPharm. Anhydrous chloroform, dimethyl sulfoxide (DMSO), and trifluoroacetic acid (TFA) were purchased from Millipore Sigma. HEPES buffer and propidium iodide (PI) were purchased from Thermo Fisher. Pierce methanol-free 16% formaldehyde was purchased from Thermo Fisher. Vectashield™ mounting medium was purchased from Vector Labs. Wheat germ agglutinin-Alexa™ Fluor 555 and Hoechst™ 33432 dyes were purchased from Thermo Fisher. 10% neutral buffered formalin, microscope slides, coverslips, slide sealer nail polish, and tissue cassettes were purchased from VWR. LabTek™ 8-chamber coverslips, Hoechst™ 33342, and fluorescently labeled wheat germ agglutinin were purchased from Thermo Fisher. DMEM (#10-013-CV) Penicillin / Streptomycin and fetal bovine serum (FBS) Coming. Primary human astrocytes (CAT #1800) and media components were purchased from ScienCell. Eagle’s Minimum Essential Media (30-2003) was purchased from ATCC.

[0083] Cell Lines

[0084] GBM6, GBM12, GBM22, and GBM39 cells were obtained from the Mayo Clinic Patient-Derived Xenograft National Resource under a Materials Transfer Agreement. Cryopreserved tumor samples were implanted into the flanks of nude mice following established protocols.40When tumors reached a diameter of 1.5 cm, cells were dissociated manually using a scalpel and syringe to break up the tissue and plated in DMEM (Coming #10-013-CV) supplemented with 2.5% fetal bovine serum and 1% pen strep. Once cells adhered to the tissue culture flasks, media was replaced with fresh DMEM containing 10% fetal bovine serum and 1% pen strep. Media was replaced twice weekly, and cells were passaged every 5-7 days at a 1:6 to 1:8 using 0.25% Trypsin-EDTA. Cells for in vitro and in vivo assays were used within 2 weeks of tumor dissociation. Primary human astrocytes (ScienCell, CAT #1800) and HCM3 microglia (ATCC) were cultured according to the manufacturers' instructions.

[0085] MMAF Conjugation

[0086] To prepare an MMAF lipid drug conjugate, DPPT (5 mg, 0.0068 mmol) and mcMMAF (6.33 mg, 0.0068 mmol) were dissolved in chloroform (total reaction volume: 1.37 mb) with 2% (v / v) triethylamine (27.38 pL) to catalyze the reaction. The mixture was stirred at room temperature for 30 minutes. The conjugation of DPPT to MMAF was monitored using reverse-phase high-performance liquid chromatography (Agilent 1260 Infinity II RP-HPLC) on a Hypersil™ GOLD™ C4 column (150 x 4.6 mm, 5 pm, Part No. 25505-154630). The mobile phase consisted of water with 0.5% trifluoroacetic acid (TFA) (solvent A) and isopropanol (solvent B), with a flow rate of 1.0 mL / min. The gradient started with 60% solvent A and 40% solvent B, held for 1 minute, followed by a ramp to 95% solvent B over 5 minutes, and maintained at 95% for an additional 10 minutes. The injection solvent was DMSO. Elution was monitored by absorbance at 220 run, with MMAF and DPPT-MMAF retention times at 3.8 and 8.0 minutes, respectively. To prepare fluorescently tagged PE-BDP Fl, the same procedure was used, replacing mcMMAF with BDP Fl- maleimide. For mass analysis, samples were injected into a Q-ToF mass spectrometer (Agilent 6545 Q-TOF LC / MS) in direct injection mode. The injection solvent was 80% methanol and 20% chloroform, with an injection volume of 1 pL. The mobile phase consisted of 80% isopropanol with 0.1% formic acid and 20% water with 0.1% formic acid at a flow rate of 0.3 mL / min using an Agilent 1260 Infinity II system. The instrument was operated in negative ionization mode, and molecular weights were analyzed using Agilent MassHunter™ software. Peaks corresponding to deprotonated molecular ions (| M-H| ) and doubly deprotonated molecular ions (| M-2H |2) were identified for DPPT, mcMMAF, and DPPT- MMAF. NP Synthesis

[0087] Fluorescent liposomes were prepared using a thin film hydration method. Briefly, cholesterol and lipids were combined in a round-bottom flask at a mol ratio of 40 DSPC: 20 DSPG: 40 Choi: 0.2 PC-BDP 650 / 665. To prepare liposome-thiol and liposome-MMAF, an additional 1% (mol / mol) of DPPT or DPPT-MMAF was added to the round-bottom flask, respectively. The lipid solution was dried at room temperature using a BUCHI RotoVap™ system until completely dry (< 50 mbar) and placed in a desiccator under vacuum overnight. The lipid film was rehydrated to a total lipid concentration of 0.5 mg / mL in a 25 mM HEPES 20 mM NaCl buffer solution and sonicated three times (1 minute on, 1 minute off) at 65 °C in a bath sonicator. Liposomes were extruded through nucleopore membranes of successively smaller diameter (400 run, 200 nm, 100 nm, 50 run) twice until a Z-average diameter of 80- 90 nm was achieved. Traces of unconjugated dye were removed by tangential flow filtration (TFF).

[0088] LbL NPs were prepared by mixing an equal volume of polyelectrolyte and NP solutions under brief sonication (3 seconds) followed by vortex mixing (3 seconds). NP solutions were prepared in deionized water (MilliQ™) at 0.5 mg / mL lipids, while polymer solutions were prepared in 50 mM HEPES 40 mM NaCl. The polymer: lipid weight equivalent for poly-L-arginine (PLR) was 0.4: 1. For co-PLE layering, the polymer solution consistent of 60% (w / w) poly-L-glutamine (PLE) and 40% (w / w) PEG-b-poly-L-glutamine (PLE-PEG), and the total polymer: lipid weight equivalent was 0.6: 1. After the adsorption of each layer, excess polymer was removed using tangential flow filtration into MilliQ™ water, and NPs were concentrated to a lipid concentration of at least 0.5 mg / mL. Following TFF purification, an aliquot of NPs was analyzed for MMAF concentration via HPLC to confirm minimal loss of the DPPT-MMAF lipid, and groups were normalized based on MMAF concentration for subsequent in vitro and in vivo assays.

[0089] Tangential Flow Filtration

[0090] NPs were purified using a Spectrum Labs KrosFlo™ II filtration system and Masterflex™ Teflon tubing. Hollow fiber filters (D02-E100-05-N for batch volumes >10 mL and C02-E100-05-N for batch volumes <10 mL), both with 100 kDa molecular weight cutoffs, were used to remove excess polyelectrolytes. For PLR purification, dedicated PLR columns were pre-treated with 1 mg / mL free PLR solution and circulated for 5-10 minutes to saturate any nonspecific electrostatic binding to the filters. For small batches, samples were concentrated to 2-3 mL (up to 2 mg / mL lipid) by circulating NP solutions through the column without any permeate solution to make up for the lost volume exiting through the waste line. For purification, MilliQ™ water was connected to the permeate for purification, and samples were circulated for 5 buffer exchanges at a flow rate of 7 mL / min. For large batches, samples were concentrated to 5 mL (up to 2 mg / mL) and purified at a 40 mL / min flow rate for 5 buffer exchanges. NP samples were kept on ice throughout the purification. Columns were backflushed to recover any sample remaining in the column and tubing.

[0091] NP Characterization

[0092] Nanoparticle hydrodynamic diameter, polydispersity index, and zeta potential were measured using dynamic light scattering (DLS) with a Malvern Zetasizer™ Advanced Pro k= 633 nm, 0 = 90°). NPs (25 pL at 0.25 mg / mL total lipid in MilliQ™ water) were mixed with 25 pL of 50 mM HEPES, 40 mM NaCl buffer and diluted with 750 pL of MillliQ™ water before being transferred to polystyrene cuvettes or DTS1070 folded capillary cuvettes. The refractive index used was 1.45, with an absorption coefficient of 0.001. For colloidal stability studies, NPs were diluted in 750 pL of artificial cerebrospinal fluid (aCSF) containing 119 mM NaCl, 2.5 mM KC1, 1.3 mM MgCh, 2 mM CaCh, 1 mM NaH2PO4, 26.2 mM NaHCO.r and 10 mM D-Glucose, all purchased from Millipore Sigma.52Solutions were supplemented with 0.02% sodium azide and incubated at 37° C overnight prior to DLS analysis. Cryo-transmission electron microscopy images were acquired using a JOEL 2100F microscope (200 kV) under cryogenic conditions.

[0093] ABD-F Thiol Detection Assay

[0094] NP samples were diluted 1: 1 into 100 mM Tris-borate-EDTA (TBE) buffer, pH 8.1- 8.5 containing 2 mM TCEP. L-cysteine was dissolved in TBE buffer and used to generate a standard curve of free thiols. In a typical reaction, 200 pL of NP or L-cysteine solution was mixed with an equal volume of 1 mM ABD-F dissolved in TBE buffer. Tubes were heated to 50 °C in a water bath for 5 minutes and then cooled on wet ice for 3 minutes. 120 pL of 100 mM HC1 was then added to the reaction mixture to stop the reaction. 150 pL of the final mixed was transferred to a black 96-well plate and fluorescence was measured using a plate reader (Tecan M200 Pro) with excitation at 389 nm and emission at 513 nm. The concentration of free thiols in the Lipo-MMAF group were normalized to the concentration of free thiols in the Lipo-thiol group to estimate the fraction of surface thiols that were consumed by the mc-MMAF. Cytotoxicity Assays

[0095] Cytotoxicity of MMAF formulations was assessed using a CellTiter-Blue™ resazurin assay (Promega). Cells were seeded in 96-well plates at a density of 10,000 cells per well in 100 pL of culture medium and allowed to adhere overnight. The next day, the media was gently aspirated and replaced with 90 pL of fresh media. Then, 10 pL of NPs (in MilliQ™ water), EGFR-MMAF (in PBS), or free MMAF (in 10% DMSO in media) stock solutions were added to each well using an Integra pipette, resulting in final well concentrations of 10% v / v water, 10% v / v PBS, or 1% v / v DMSO, respectively. Untreated control wells received equivalent volumes of water, PBS, or DMSO corresponding to each treatment condition. The cells were incubated for 72 hours, after which 20 pL of CellTiter-Blue™ reagent was added to each well and incubated for 1 hour. Viability was assessed by measuring fluorescence (excitation at 560 nm and emission at 590 nm) on a plate reader (Tecan M200 Pro) and normalized to untreated controls. MG132 (40 pM) was used as a positive control for cell death.

[0096] Flow Cytometry Uptake Study

[0097] The cellular uptake of BDP Fl -tagged liposome and UbU NPs were assessed using a BD USR II Flow Cytometer with a high-throughput sampler. GBM22 cells were seeded on a 96- well plate at a density of 10,000 cells per well in 100 pU culture media and allowed to adhere overnight. NP stock solutions were prepared at 0.1 mg / mU in MilliQ™ water and 5 pU was transferred to each well for an incubation period of 4 or 24 hours. Cells were then washed three times with PBS and detached with 25 pU of trypsin-EDTA. 200 pL FACS buffer (PBS containing 1% bovine serum albumin and 1 mM EDTA) with 1 pg / mL propidium iodide (Thermo Fisher) was added to each well, and the cells were pipetted vigorously 3-4 times to achieve a single-cell suspension. Propidium iodide staining was detected on the PE-Texas Red™ channel (ex. 488 em. 610 / 20), and BDP 650 / 665 tagged NPs were detected on the APC channel (ex. 640 em. 670 / 30). Data were analyzed using FlowJo™ (version 10), and gates for single cells, live cells, and NP -positive cells were applied using untreated samples.

[0098] Confocal Microscopy of NP-treated cells

[0099] LabTek #1.5 borosilicate 8-well chamber slides were coated with 300 pL of 50 pg / mL rat tail collagen (Millipore Sigma) in 0.02N acetic acid for 5 minutes at room temperature. The slides were then washed once with PBS and air-dried for 5 minutes. GBM22 cells (10,000 per well) were seeded in 300 pL of media and allowed to adhere overnight before treatment with 5 pg / mL fluorescent NPs for 24 hours. After treatment, cells were carefully washed three times with ice-cold HBSS and incubated with 2 pg / mL wheat germ agglutinin-Alexa™ Fluor 555 (Thermo Fisher) in ice-cold HBSS for 2 minutes. Excess wheat germ agglutinin was removed with a single wash of ice-cold PBS, followed by two additional PBS washes (5 minutes each). Cells were fixed with 4% paraformaldehyde (Pierce) for 15 minutes at room temperature, washed three more times with PBS, and then stained with 4 pM Hoechst™ 33432 (Thermo Fisher) for 5 minutes at room temperature. Finally, cells were imaged using a Leica SP8 confocal microscope with a 63x or lOOx oil immersion objective, and images were pseudo colored using FIJI. Colocalization analysis was performed using the JaCoP image J™ plugin53to calculate the Mander’s coefficient for DPPT-MMAF signal overlap with LbL NPs. Images were thresholded using the Costes Automatic Thresholding algorithm built into JaCoP™.

[0100] Animal Studies

[0101] All animal experiments were approved by the Massachusetts Institute of Technology Committee on Animal Care (CAC, protocol number 2207000386) and were conducted under the oversight of the Division of Comparative Medicine (DCM). NCR / Nude mice were purchased from Taconic and housed in cages with a maximum of five animals per cage, under controlled temperature (25 °C), 12-hour light-dark cycles, and provided with free access to food and water. Both male and female mice were used for all experiments, and the mice were 7-10 weeks at the time of the experiment. Group sizes were determined using MANOVA for repeated measures (alpha = 0.05, power = 0.95) based on effect sizes and standard deviations from pilot experiments.

[0102] Orthotopic Tumor Inoculation

[0103] Mice were anesthetized with a mixture of ketamine (100 mg / kg) and xylazine (10 mg / kg) administered intraperitoneally. Mice were transferred to a 37 °C heating pad, and ophthalmic ointment was applied using a sterile Q-tip™. Anesthesia was confirmed using a toe pinch, and Buprenorphine SR (1 mg / mL) was administered subcutaneously. The skull was swabbed with alternating Betadine and 70% ethanol three times, and a midline skin incision approximately 1 cm in length was made using a sterile scalpel. The periosteum was swabbed with a sterile Q-tip™ to expose the bregma, and a lidocaine 2.5% / prilocaine 2.5% cream was applied to the ears before placement on a stereotaxic frame (Stoelting). A small burr hole located 1.8 mm right and 0.8 mm anterior to the bregma was drilled using a 1 mm steel drill bit. The Stereodrive™ software was used to align the needle with the burr hole and lower it to a depth of 3.5 mm from the skull surface. 100,000 GBM12 cells in 3 pL were administered intracranially over 3 minutes using a 10 pL Hamilton syringe fitted with a 34 G stainless steel needle. The needle was left for 2 minutes to equilibrate in the brain prior to infusion of cells. After an additional 2 minutes of equilibration after cell infusion, the needle was raised at a rate of 1 mm / min. The incision was closed using 5-0 monocryl sutures (Ethicon) and sealed with tissue glue. Animals were placed in a heated cage for post-op recovery and monitored daily for the first 4 days, then twice weekly until the experimental endpoint was reached.

[0104] Convection-Enhanced Delivery

[0105] CED was performed on tumor-bearing mice 8 days after tumor inoculation in the same coordinates. To account for potential experimental variability across different days, each day of CED treatments included a mixture of different treatment groups. CED was performed using the same protocol for tumor inoculations with several modifications. NPs or control solutions were diluted with sterile dextrose solution prior to injection to achieve 5% (w / w) dextrose. DLS analysis confirms that both Lipo-MMAF and LbL-MMAF maintain their <100 run Z-average diameter when suspended in 5% dextrose. Approximately 7 pL NP, ADC, or control solution was drawn into a 10 pL Hamilton syringe fitted with a custom- made 34 G stainless steel needle protruding 1 mm from a 27 G blunt-tipped needle.54The Stereodrive™ software was used to align the needle with the burr hole and lower it to a depth of 3.5 mm from the skull surface. The needle was left for 2 minutes to equilibrate in the brain prior to infusion of either a 5% dextrose control, MMAF, Lipo-MMAF, LbL-MMAF, or EGFR-MMAF at a matched MMAF dose of 3.4 pg / kg. The carrier fluid for all samples was 5% dextrose in sterile water. A motorized injection robot (Stoelting) was used to infuse at a ramped injection protocol: 1 pL at 0.2 pL / min, 2 pL at 0.5 pL / min, and 3 pL at 0.8 pL / min.

[0106] Magnetic Resonance Imaging

[0107] Magnetic Resonance Imaging (MRI) was performed in vivo on a 7T MRI operated by Bruker AV4 NeoBioSpecT70-20USR™ console, equipped with a 114mm 660mT / m actively shielded gradient and a QSN075 / 040 RF coil (Bruker BioSpin™, Rheinstetten, Germany). Mice were anesthetized by inhalation of 2.5% isoflurane and maintained on 2-2.5% isoflurane throughout data collection. Body temperature and respiration rate were monitored by SAII monitoring and gating system (Small Animal Instruments Inc., Stony Brook). Anatomical brain images were collected and reconstructed within Bruker Paravision PV360 v2.0. T2 Weighted images were obtained using the TurboRARE™ protocol with

[0108] TR / TE=3000 / 25 ms, number of averages=4, and RARE factor of 8. Coronal datasets were obtained with the geometric parameters of 256x256 matrix, field of view (FOV)=20x20mm2, interleaved number of slices=20, no gap and slice thickness=0.5mm. Images were converted to DICOM format and exported to 3D slicer for analysis.

[0109] Brain Histology and Cryofluorescence

[0110] For immunofluorescence studies, brains were removed and fixed in 10% formalin overnight at room temperature. The olfactory bulb and cerebellum were removed, and the remaining brain tissue was fixed in fresh 10% formalin for an additional hour. Tissues were cryoprotected in 15% (w / w) sucrose in PBS overnight, followed by 30% (w / w) sucrose in PBS for an additional 24 hours. Brains were embedded in optimal cutting temperature compound (TissueTek™), placed in a 15 mm x 15 mm tissue mold, and frozen on dry ice. Frozen tissue was stored at -80 °C prior to sectioning. Serial sections with a thickness of 10 pm were cut using a cryostat (Leica), sequentially adding one section to a slide, with 10 slides total. This process was repeated 6 times, resulting in 10 slides, each with six brain sections spaced 100 pm apart. Two sets of 10 slides were collected for each brain, to cover the entire region of NP distribution.

[0111] For visualization of NP distribution and cell nuclei alone, slides with frozen sections were dried at room temperature for 30 minutes in the dark and rehydrated in PBS for 10 minutes. Slides were stained with Hoechst™ 33432 (10 pM, Thermo Fisher) in PBS for 10 minutes. Sections were washed three times for 5 minutes each in PBS and mounted with Vectashield™ Vibrance™ mounting media (Vector Labs). For immunofluorescence, slides were dried at room temperature for 30 minutes, rehydrated in PBS for 10 minutes, and permeabilized with 0.2% triton-X in PBS (Millipore Sigma) for 15 minutes. Slides were washed three times for 5 minutes each in PBS and blocked using 10% normal goat serum (Cell Signalling Technologies) and 0.1% (w / w) Tween-20 (Millipore Sigma) in PBS. For EGFR-MMAF-treated samples, goat anti-human IgG- Alexa™ Fluor 647 (Invitrogen A21445) at a 1:400 dilution in PBS with 1% (w / w) bovine serum albumin was added and incubated for 1 hour at room temperature, and samples were washed three times with PBS for 5 minutes each. For all samples, primary antibodies were diluted in PBS with 1% (w / w) bovine serum albumin and 0.1% (w / w) Tween 20™ at the following dilutions: vimentin- AF488 (Abeam abl95877) 1:300, MMAF (Levena Biopharma LEV-PAF 1-100) 1:200, cleaved caspase 3 (Cell Signalling Technology #9661) 1:300, NeuN (Abeam abl04225) 1:500, Ibal (Abeam abl78846) 1:500, EGFR (Cell Signalling Technology #2232S) 1:200. Slides were incubated with the primary antibodies overnight at 4 °C, then washed three times with PBS for 5 minutes each. The anti-rabbit AF488-plus secondary antibody (Invitrogen A32731) was diluted 1 : 1000 in PBS with 1% BSA and added to the slides for a 1-hour incubation at room temperature. Slides were washed three times with PBS for 5 minutes each and stained with DAPI (5 pg / mL, Thermo Fisher) in PBS for 10 minutes. Sections were washed three times for 5 minutes each in PBS and mounted with ProLong™ Gold Antifade Mounting Medium. Slides were imaged using confocal microscopy (Leica SP8) and pseudo colored using FIJI.

[0112] For quantitative analysis of cleaved caspase-3 staining, images were converted into 8- bit TIFF fdes and imported into QuPath™ version (v)0.5.1.55To calculate the percentage of total cells in the tumor, a region of interest (ROI) was manually drawn around the tumor area, identified by the presence of hVimentin staining. Nuclei were detected using the “Cell Detection” feature, with the DAPI channel as the detection channel, an intensity threshold of 5, and a cell expansion of 5 pm. A single measurement classifier was applied to determine whether each cell was positive or negative for cleaved caspase-3, with a threshold set to 10 on the cleaved caspase-3 channel. To calculate the percentage of apoptotic cells that were also tumor cells, the “Cell Detection” feature was applied to the cleaved caspase-3 channel with an intensity threshold of 10 and a cell expansion of 0 pm. A single measurement classifier then determined whether each apoptotic cell was positive or negative for hVimentin, using a threshold of 30 on the hVimentin channel.

[0113] Toxicity Study

[0114] To evaluate the toxicity of different MMAF formulations, healthy nude mice were infused with 5% dextrose, MMAF, Lipo-MMAF, LbL-MMAF, EGFR-MMAF, and an LbL- MMAF vehicle using the same dose and procedures as the therapeutic efficacy study. Mice were weighed daily and monitored for signs of behavioral neurotoxicity. Blood samples were collected via submandibular bleed on day 3 days, 7 days, and long-term (3 weeks) for complete blood count analysis. On day 7, additional blood was collected via a cardiac puncture for a liver enzyme panel. All blood tests were performed by MIT's Division of Comparative Medicine Comparative Pathology Lab and compared to the normal ranges for mice. Brains were removed and fixed in 10% formalin overnight at room temperature, and a coronal cut was made using a razor blade to expose two surfaces at the injection site. The tissue was fixed in fresh 10% formalin for an additional hour and placed cut-side up in a tissue cassette. The tissue was embedded in paraffin, and then 5 pairs of two 4 pm sections were taken at intervals of 50 pm. As a result, the total depth of the examined or viewed tissue was 500 pm. Slides were stained with H&E, and a certified pathologist blindly evaluated the tissue sections.

[0115] Statistical Analysis

[0116] All statistical analysis was performed using GraphPad Prism 9™. Information on statistical tests is provided in the associated figure caption.

[0117] Example 1. Synthesis of lipid drug conjugate

[0118] An illustrative lipid drug conjugate was prepared using 1,2-dipalymitoyl-sn-glycero- 3 -phosphothioethanol (DPPT or alternatively, 16:0 PE-thioethanol) and monomethyl auristatin F (MMAF). The drug conjugate was synthesized prior to nanoparticle formulation. Briefly, for the synthesis a light molar excess of 16:0 PE-thioethanol (DPPT) was mixed with maleimidocaproyl-MMAF (mc-MMAF) in chloroform with 2% (v / v) triethylamine for 1 hour at room temperature (Figure 2A). To confirm the 1: 1 stoichiometry of the conjugation reaction, the consumption of mc-MMAF was monitored and confirmed by HPLC (Figure 2B). As shown, when mc-MMAF was reacted with an initial 0.25 molar equivalents of DPPT, the peak area associated with mc-MMAF decreased by approximately 25%. Upon addition of the remaining molar equivalents of DPPT (approximately 0.75 eqs), the mc- MMAF HPLC peak disappeared, indicating complete consumption of the mc-MMAF moiety. These reaction conditions allow for the mc-MMAF reactant to be fully consumed, and no further purification step was needed to remove any unreacted drug precursor (mc-MMAF).

[0119] Example 2. Liposome generation using lipid drug conjugate

[0120] The strategy of tethering the active drug (e.g., mc-MMAF) to a saturated lipid (e.g., DPPT) allows for the straightforward incorporation of the resulting lipid drug conjugate (e.g., DPPT-MMAF) into lipid solutions during the preparation of a lipid film and resulting liposomes. To understand the effect that the incorporation of lipid drug conjugate (e.g., DPPT-MMAF from Example 1) has on liposome formation and formulation, three versions of saturated liposomes were prepared based on known formulations (see, e.g., Pickering et al. 2023). In this evaluation protocol the three variations include: liposomes, liposomes containing 1% (mol / mol) DPPT, and liposomes containing 1% (mol / mol) lipid drug conjugate (DPPT-MMAF) (see, Figure 3A). Following extrusion through 50 nm fdters, all three liposomes had a Z-average diameter of 85-90 nm with low polydispersity indices below 0.2, as well as zeta potentials of -40 to -50 mV, with no significant differences between groups (Figure 3B, C). A thiol detection assay confirmed the presence of thiol groups from the presence of DPPT, whereas approximately 94% of the thiols were consumed by mc- MMAF in the MMAF-conjugated formulation (Figure 3D). While this measurement confirms a slight excess of DPPT in the conjugation reaction, the presence of a small amount of free thiol groups is not expected to have substantial affect the properties of the liposomes.

[0121] Example 3. LbL assembly

[0122] The MMAF conjugated liposomes generated in Example 2 were functionalized with LbL assembly generally following the protocol as described in Pickering et al. (2023) (Figure 3E). In brief overview of the preparation of LbL NPs, an equal volume of the polyelectrolyte and NP solutions were mixed under brief sonication (e.g., about 3 seconds) and vortex mixed (e.g., about 3 seconds). Polyelectrolyte solutions were prepared in a buffer of 50 mM HEPES and 40 mM NaCl. NP solutions were prepared in deionized water (Milli- Q) at 0.5 mg / mL lipids. The weight equivalent of polyelectrolyte to liposome core was 0.6x for poly-L-arginine (PLR) and l.Ox for poly-L-glutamine (PLE) and PEG-6 -poly-L- glutamine (PLE-PEG). The same procedure was used for co-layering the anionic layer with PLE-PEG, except the polyelectrolyte solution contained a mixture of the two polyelectrolytes (PLE and PLE-PEG), with a total weight equivalent of polyelectrolyte to liposome core of 1.0. After the adsorption of each layer, any excess polymer was removed using tangential flow filtration and concentrated to a range of between 0.5 and 1 mg / mL.

[0123] Nanoparticles displayed controlled growth of 5-10 nm with the addition of each layer, with complete charge reversal, indicating that the presence of covalently attached DPPT- MMAF does not interfere with the layering process (Figure 3F,G). Advantageously, the generated MMAF-conjugated co-PLE LbL NPs maintained their Z-average diameter (<100 nm) when incubated in artificial cerebrospinal fluid at 31° C overnight, which indicates a high colloidal stability that allows for the particles to penetrate the narrow pores in target tissues and organs such as, for example, the brain (Figure 3H). Thus, incorporating the lipid drug conjugate (e.g., 1% DPPT-mcMMAF) does not have any substantial effects on the characteristics of resulting liposomes or the LbL functionalization process. Example 4. Activity of LbL drug conjugate nanoparticles a. In vitro toxicity.

[0124] The efficacy of the LbL-MMAF nanoparticles (NPs) generated in Example 3 was tested against glioblastoma by first assessing the in vitro cytotoxicity on patient-derived xenografts, including three EGFR-mutant glioblastomas (GBM6, GBM39, and GBM12) and one EGFR-wild type (GBM22), grown from mice flank tumors. For the in vitro assays, cells were exposed to various concentrations of free MMAF, liposome-MMAF, LbL-MMAF, or an anti-EGFR-MMAF ADC for 72 hours, with cell viability measured by a resazurin metabolic assay (Figure 4A i-iv). As observed, free MMAF showed high IC50 values (e.g. ranging from 0.2 - 0.9 pM), while the liposome-MMAF had substantially greater efficacy (IC50s in a range of about 2-14 nM), suggesting the benefit of cytoplasmic delivery via nanoparticle internalization (Figure 4B). Further functionalization of the liposome with Co- PLE enhanced potency, reducing IC50 values in corresponding cell types by about 2- to 4- fold when compared to liposome-MMAF (e.g., IC50s ranging from about 1-3.5 nM). Thus, both NP formulations outperformed the EGFR-MMAF ADC across all cell lines, and particularly in the EGFR-wild type GBM22, suggesting that LbL NPs may have broader potential in targeting various GBM cells regardless of their mutation status.

[0125] Cytotoxicity of the NPs against normal, healthy cells was evaluated using primary human astrocytes and HCM3 immortalized microglia (Figure 4 A v-vi). While both the liposome-MMAF and LbL-MMAF showed cytotoxicity, LbL-MMAF was less potent against healthy cells relative to the tested cancer cells. No cytotoxicity against the normal cells was observed at any tested concentration for either the free MMAF or the EGFR-MMAF ADC, suggesting that incorporating targeting chemistries on nanoparticles can reduce off-target cell uptake. LbL functionalization can significantly reduce uptake and internalization of such modified liposomes in healthy cells (e.g., astrocytes, oligodendrocytes, and other neural cells) when compared to unmodified liposomes (see, e.g., Pickering et al., 2023). b. In vivo efficacy.

[0126] A tumor treatment experiment was used to evaluate the efficacy of the LbL-MMAF NPs generated in the above Examples in immunocompromised mice bearing intracranial GBM12 glioblastoma tumors (Figure 5A). Briefly, seven days after tumor inoculation, mice were randomized into treatment groups testing LbL-MMAF NPs or controls: 5% dextrose vehicle, free MMAF, and liposome-conjugated MMAF. As an additional control representing an alternate delivery strategy for MMAF, an antibody-drug conjugate of EGFR-MMAF was tested (Figure 5B). GBM12 has an EGFR mutation but has previously been shown to develop resistance to EGFR-targeting ADCs, motivating the application of nanoparticlebased MMAF formulations that are not dependent on the expression of a single protein (Marin, B.-M., et al., “Heterogeneous Delivery across the Blood-Brain Barrier Limits the Efficacy of an EGFR-Targeting Antibody Drug Conjugate in Glioblastoma”. Neuro-Oncol. 2021, 23 (12), 2042-2053.).

[0127] Mice were treated with a single CED infusion of LbL-MMAF NPs at a dose of 0.3 mg / kg lipid. All control groups except dextrose were delivered at a matched MMAF dose. T2 -weighted MRI scans confirmed delayed tumor growth in animals receiving either liposome-conjugated MMAF, LbL-MMAF NPs, or EGFR-MMAF (Figure 5C, D). CED treatment did not result in significant weight loss or symptoms of neurological damage in any groups until the tumors had progressed, indicating that the treatment was well tolerated (Figure 5E). While mice treated with a 5% dextrose control infusion had a median survival of 31 days, LbL-MMAF NPs significantly prolonged the median survival to 85 days (Figure 5F). This was longer than the median survival times of both liposome-conjugated MMAF and EGFR-MMAF, which had median survival times of 44 and 64 days, respectively. c. Biological distribution.

[0128] The distribution of LbL-MMAF NPs was evaluated in GBM12 tumors through cryofluorescence microscopy of brain tissue sections 24 hours after CED (Figure 6A-B). A merged image (left panel) of individual stained images including specific dyes (Figure 6A). The general DNA binding dye DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) is shown in Figure 6A (left-center panel) and Figure 6B (left panels). Fluorescently tagged nanoparticle lipids (Figure 6A, right-center panel, shown in red) can be seen to penetrate and localize to GBM12 tumor tissue, as stained with anti-human vimentin (Figure 6A, center panel, shown in green). Notably, the distribution of the MMAF payload itself was visualized with an anti-MMAF antibody (Figure 6A, right panel, shown in cyan), showing a similar distribution to the nanoparticle core. Previous studies of CED of both small molecules and proteins have shown rapid brain clearance within 24 hours (Pickering A. J., et al., 2023), suggesting that LbL-MMAF is a promising way to extend the local retention and delivery of MMAF to glioblastoma cells. Staining for apoptosis using cleaved-caspase 3 confirms the apoptotic activity of MMAF in the tumor 24 hours after delivery of MMAF (Figure 6B). Minimal cleaved-caspase 3 signal was observed in mice receiving a dextrose vehicle injection, confirming that the cytotoxicity was due to the LbL-MMAF. Ten days after LbL- MMAF delivery to GBM12 tumors, neuron staining with NeuN reveals no widespread neuron loss throughout the treated hemisphere of the brain, indicating successful tumor regression and minimal neurotoxicity associated with treatment (Figure 6C).

[0129] Example 5. MMAF carrier distribution, activity, and retention impact anti-GBM efficacy in vivo

[0130] We further investigated the tissue and cellular interactions of LbL-MMAF and control formulations to elucidate the mechanisms underlying their therapeutic efficacy. A separate cohort of GBM12 tumor-bearing mice was treated with a CED single infusion of either 5% dextrose, Lipo-MMAF, LbL-MMAF, or EGFR-MMAF (n=4 per condition). Brains were harvested and sectioned for cryo-fluorescence microscopy at 24 hours, 5 days, and 10 days post-CED to assess MMAF and carrier distribution. Liposome and LbL NP carriers were visualized via covalently-conjugated fluorescent lipids (DPSC-BDP 650), MMAF via an anti- MMAF antibody, and EGFR-MMAF ADC carriers via an anti-human IgG antibody. At 24 hours post-CED, similarly sized, dense tumors were observed across all treatment groups (Figure 7A). Lipo-MMAF displayed limited penetration from the infusion site, with most NP fluorescence concentrated in the tumor core, whereas LbL-MMAF exhibited extensive distribution throughout the entire tumor area. In both cases, there was substantial overlap between NP fluorescence and MMAF distribution, detected using an anti-MMAF antibody, suggesting minimal drug leakage or lipid exchange from the nanoparticle depot. EGFR- MMAF spread throughout the entire hemisphere, as expected for protein formulations due to their smaller size compared to liposomes. The anti-EGFR IgG signal colocalized with MMAF throughout the hemisphere, with a more concentrated signal in the tumor bulk, a region of high EGFR overexpression. The increased intensity of the MMAF signal within the tumor compared to the EGFR-MMAF ADC carrier may be due to signal amplification, as MMAF was detected with both a primary and secondary antibody, whereas the ADC carrier was detected with only a secondary anti-human antibody. The carrier distribution patterns of Lipo-MMAF, LbL-MMAF, and EGFR-MMAF were consistent across four biological replicates. Consistent with survival data with these agents, the complete tumor coverage achieved with LbL-MMAF and EGFR-MMAF enabled broader drug distribution, leading to delayed tumor regrowth and improved survival compared to Lipo-MMAF.

[0131] After investigating trafficking of the carrier and drug, we next turned attention to pharmacodynamic effects of MMAF in the same samples. MMAF exerts its therapeutic effect by disrupting mitosis in rapidly dividing cells and inducing apoptosis. We confirmed the apoptotic activity of the MMAF formulations by staining for cleaved caspase-3 (cCasp-3), a marker of apoptosis (Figure 7B). LbL-MMAF induced a significantly higher fraction of cCasp-3+ cells (7.2%) within the tumor area compared to Lipo-MMAF (1.2%) and EGFR- MMAF (0.5%) (Figure 7C). The superior performance of LbL-MMAF NPs may be attributed to the increased anti-GBM potency revealed with in vitro cell viability assays. The broad initial distribution of EGFR-MMAF may also have resulted in a dilution effect, diminishing the local drug concentration within the tumor and consequently lowering its potency. In the case of Lipo-MMAF, the reduced efficacy may be further explained by cellular distribution studies, which indicated that non-functionalized liposomes tend to accumulate in macrophages and microglia - cell types less susceptible to microtubule inhibitors. We assessed the specificity of tumor cell killing by analyzing the overlap between the cCasp-3 signal and human vimentin (hVim), a marker of human PDX tissue. While only 49. 1% of apoptotic cells in the Lipo-MMAF group were hVim+, 72.4% and 74.5% of apoptotic cells in the LbL-MMAF and EGFR-MMAF groups, respectively, were hVim+ (Figure 7D). This indicates significantly higher tumor-specific killing by LbL-MMAF compared to Lipo-MMAF. Surprisingly, EGFR-MMAF did not demonstrate greater tumor specificity than LbL-MMAF despite its high specificity for EGFR+ cells. Importantly, the distribution of the cCasp-3 signal following LbL-MMAF treatment was confined to the tumor area; no apoptosis was detected in other brain regions.

[0132] Given the sustained therapeutic benefit observed from a single administration of LbL- MMAF and EGFR-MMAF, we next investigated the local retention of MMAF and their respective drug carriers at different timepoints. Five days post-CED, LbL NP fluorescence remained localized to the tumor region (Figure 7E, top row). Ten days post-CED, very few tumor cells were present, and the NP fluorescence had notably decreased (Figure 7E, bottom row). Co-localization between NP fluorescence and MMAF signal at both time points suggests that the drug remains associated with the nanoparticles throughout their residence time in the brain. Despite the potential for thiol-maleimide bond reversal in vivo, these results indicate prolonged drug retention within the tumor and no detectable spread to other brain regions. The retention of LbL NPs in treated tumors contrasts with previous studies in healthy brain tissue, where LbL NPs without MMAF remained localized at the injection site for at least 21 days. We hypothesize that the reduced NP retention in the tumor model could be due to microglia and macrophage-mediated phagocytosis and clearance of dead tumor tissue. Supporting this, we observed co-localization between LbL NP fluorescence and Ibal, a microglia marker, at the tumor site ten days post-CED (data not shown). At this time point, there was no widespread neurotoxicity throughout the treated hemisphere, with only a limited region of neuron loss along the needle tract and collapsed tumor tissue.

[0133] In EGFR-MMAF -treated groups, human IgG staining was absent in the tumor at both the 5- and 10-day time points, though MMAF staining was detected and colocalized with the tumor signal at day 5 (Figure 7F, top row). By day 10, however, MMAF was no longer detectable, and a sparse population of tumor cells persisted, and MMAF fluorescence was undetectable above background (Figure 7F, bottom row). Unlike LbL NPs, ADCs have limited adhesion to the extracellular matrix, depending instead on sustained high levels of surface EGFR expression to remain associated with tumor tissue. Notably, EGFR overexpression persisted at day 10, indicating that adaptive resistance did not account for the reduced efficacy observed in this group. Instead, the diminished retention of the ADC and the subsequent depletion of MMAF within the tumor likely explain the tumor regrowth. Overall, these findings demonstrate that the enhanced tumor penetration and sustained drug retention achieved by LbL-MMAF NPs result in superior tumor-specific killing and prolonged therapeutic efficacy, underscoring the importance of carrier design in optimizing the distribution and potency of drug formulations in GBM treatment.

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Claims

We claim1. A particle, comprising:(a) a liposomal lipid bilayer comprising a negatively charged outer surface and a lipid-drug conjugate, wherein the lipid-drug conjugate comprises a covalent bond between the lipid and the drug;(b) a first layer comprising a cationic polymer, wherein the cationic polymer is non-covalently associated with the negatively charged outer surface of the liposome; and(c) a second layer, comprising a combination of an anionic polymer and a polyethylene glycol modified anionic polymer, wherein the second layer is non-covalently associated with the first layer.

2. The particle of claim 1, wherein the particle has a ratio of about 0.4 to about 0.8 weight equivalent of cationic polymer to liposome lipid bilayer, or a ratio of about 0.6 weight equivalent of cationic polymer to liposome lipid bilayer, wherein the ratio is sufficient to yield a zeta potential greater than 30 mV.

3. The particle of claim 1 or 2, wherein the particle has a ratio of about 0.5 to about 1.5 weight equivalents of combined anionic polymer and polyethylene glycol modified anionic polymer to liposome lipid bilayer, or a ratio of about 0.8 to about 1.2 weight equivalent of combined anionic polymer and polyethylene glycol modified anionic polymer to liposome lipid bilayer, or a ratio of about 1.0 weight equivalent of combined anionic polymer and polyethylene glycol modified anionic polymer to liposome lipid bilayer, wherein the ratio is sufficient to yield a zeta potential less than -30 mV.

4. The particle of any one of claims 1-3, wherein the combined anionic polymer and polyethylene glycol modified anionic polymer comprises a ratio of about 70:30 to about 40:60 weight equivalents of anionic polymer to polyethylene glycol modified anionic polymer, or a ratio of about 60:40 to about 50:50 weight equivalents of anionic polymer to polyethylene glycol modified anionic polymer or ratio of about 60:40 weight equivalents of anionic polymer to polyethylene glycol modified anionic polymer.

5. The particle of any one of claims 1-4, wherein the liposomal lipid bilayer comprises DPPT (l,2-dipalymitoyl-sn-glycero-3-phosphothioethanol); cholesterol; DSPC (1,2- distearoyl-sn-glycero-3-phosphocholine); DSPE (l,2-Distearoyl-sn-glycero-3-phosphoethanolamine); 18:0 propargyl PC (l,2-distearoyl-sn-glycero-3-phosphocholine (N- propynyl)); 16:0 azidocaproyl PE (l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(6- azidohexanoyl)); 18:0 azidoethyl PC (l,2-distearoyl-sn-glycero-3-phosphocholine (N- azidoethyl)); 16:0 DBCO PE (l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- dibenzocyclooctyl); DPPC (l,2-dipalmitoyl-glycero-3-phosphocholine); DMPC (1,2- dimyristoyl-sn-glycero-3-phosphochline); POPC (1 -palmitoyl -2 -oleoyl-glycero-3- phosphocholine); or DSPG (l,2-Distearoyl-sn-glycero-3-phosphoglycerol), or derivatives thereof, or any combination of one or more thereof.

6. A particle, comprising:(a) a liposomal lipid bilayer comprising l,2-dipalymitoyl-sn-glycero-3- phosphothioethanol (DPPT) conjugated covalently to a drug;(b) a first layer comprising poly-L-arginine (PLR), wherein the PLR is non- covalently associated with the negatively charged outer surface of the liposome; and(c) a second layer, comprising a combination of poly-L-glutamic acid (PLE) and poly-L-glutamate-b-polyethylene glycol (PLE-PEG), wherein the second layer is non- covalently associated with the first layer.

7. The particle of claim 6, wherein the particle has a ratio of about 0.4 to about 0.8 weight equivalent of PLR to liposome lipid bilayer, or a ratio of about 0.6 weight equivalent of PLR to liposome lipid bilayer.

8. The particle of claim 6 or 7, wherein the particle has a ratio of about 0.5 to about 1.5 weight equivalents of combined PLE and PLE-PEG to liposome lipid bilayer, or a ratio of about 0.8 to about 1.2 weight equivalent of combined PLE and PLE-PEG to liposome lipid bilayer, or a ratio of about 1.0 weight equivalent of combined PLE and PLE-PEG to liposome lipid bilayer.

9. The particle of any one of claims 6-8, wherein the PLE-PEG comprises a ratio of about 70:30 to about 40:60 weight equivalents of PLE:PEG, or a ratio of about 60:40 to about 50:50 weight equivalents of PLE:PEG or ratio of about 60:40 weight equivalents of PLE:PEG.

10. The particle of any one of claims 1-9, wherein the therapeutic comprises one or more therapeutic selected from the group consisting of cytotoxins (including but not limited to dolastatins (including but not limited to dolastatin 10), auristatins (including but not limited to monomethyl auristatin E (MMAE), and monomethyl auristatin F (MMAF)), maytansines (including but not limited to ansamitocin, mertansine, and ravtansine), calicheamicins, and toxoids), angiogenesis inhibitors (including but not limited to axitinib, bevacizumab, cabozantinib, everolimus, lenalidomide, lenvatinib mesylate, pazopanib, ramucirumab, regorafenib, sorafenib, sunitinib, thalidomide, vandetanib, and ziv-aflibercept), anthracyclines (including but not limited to daunorubicin, doxorubicin, and epirubicin), immune checkpoint inhibitors (including, but not limited to, pembrolizumab, nivolumab, and cemiplimab as anti-PD-1 antibodies, ipilimumab as an anti-CTLA-4 antibody, and atezolizumab, avelumab, and durvalumab as anti-PD-Ll antibodies), microtubule stabilizers (including by not limited to docetaxel or paclitaxel), and other cancer growth inhibitors including but not limited to tyrosine kinase inhibitors (including but not limited to alectinib, brigatinib, ceritinib, crizotinib, entrectinib, lorlatinib, ALK, I, bosutinib, dasatinib, imatinib, nilotinib, ponatinib, afatinib, dacomitinib, erlotinib, gefitinib, lapatinib, neratinib, osimertinib, vandetanib, gilteritinib, midostaurin, erdafitinib, ruxolitinib, larotrectinib, axitinib, carbozantinib, lenvatinib, pazopanib, regorafenib, sorafenib, sunitinib, dabrafenib, encorafenib, vemurafenib, acalabrutinib, ibrutinib, binimetinib, cobimetinib, trametinib, abemaciclib, palbociclib, ribociclib, or eCF506), proteasome inhibitors (including but not limited to ortezomib, carfizomib, ixazomib, delanzomib, oprozomib, and marizomib), mTOR inhibitors (including but not limited to everolimus, sirolimus, temsirolimus, everolimus, sirolimus, sirolimus protein-bound, and everolimus), PI3K inhibitors (including but not limited to copanlisib, alpelisib, idelalisib, duvelisib and umbralisib), histone deacetylase inhibitors (including but not limited to vorinostat, romidepsin, panobinostat, and belinostat), and Hedgehog pathway blockers (including but not limited to vismodegib, sonidegib, and glasdegib), derivatives thereof, combinations thereof, and / or pharmaceutically acceptable salts thereof.

11. The particle of claim 10, wherein the therapeutic comprises maleimidocaproyl- monomethyl auristatin F (me -MMAF).

12. The particle of claim 11, wherein the particle comprises up to 1.0 mol % DPPT- MMAF of liposome lipid bilayer.

13. The particle of any one of claims 1-12, wherein the particle has a diameter range between about 50 nm to about 500 nm, or between about 80 to about 120 nm, or about 90 to about 100 nm.

14. A pharmaceutical composition, comprising(a) a plurality of the particles of any one of claims 1-13; and(b) a pharmaceutically acceptable carrier.

15. The pharmaceutical composition of claim 14, wherein the pharmaceutical composition is formulated for intravenous administration.

16. The pharmaceutical composition of claim 14, wherein the pharmaceutical composition is formulated for convection-enhanced delivery (CED).

17. A method for treating a cancer in the brain, comprising administering to a subject having a cancer in the brain the particle or pharmaceutical composition of any one of claims 1-16 in an amount effective to treat the cancer.

18. Particle or pharmaceutical composition of any one of claims 1-16 for use in treating a cancer in the brain.

19. The method or use of claim 17 or 18, wherein the particle comprises MMAF, or a pharmaceutically acceptable salt thereof.

20. The method or use of any one of claims 17-19, wherein the cancer in the brain is a glioma or a glioblastoma.

21. The method or use of any one of claims 17-20, wherein the particle or pharmaceutical composition is administered intravenously.

22. The method or use of any one of claims 17-20, wherein the particle or pharmaceutical composition is administered by convection-enhanced delivery (CED).

23. The method or use of any one of claims 17-22, wherein the subject is a human.

24. A method for making the particle of any one of claims 1-14, comprising generating the lipid-drug conjugate by covalently binding the lipid to the drug; reacting the lipid-drug conjugate with a lipid under conditions adequate to form a liposome; electrostatically attaching the cationic polymer of the first layer to the liposome; electrostatically attaching the second layer, comprising a combination of an anionic polymer and a polyethylene glycol modified anionic polymer to the first layer.

25. The method of claim 24, wherein the lipid-drug conjugate comprises DPPT- mcMMAF.

26. The method of 25, further comprising loading an additional therapeutic into the liposome aqueous core by adding the therapeutic to a dried lipid film before rehydration of the lipid film in water to form the particle, and loading the therapeutic into the liposome lipid bilayer by adding it to the lipid film before liposome rehydration.

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