Hybrid lipid nanoparticles containing inorganic particles and a drug of interest

Abstract

The present application relates to lipid nanoparticles comprising helper lipids and ionizable lipids, at least one lipid layer surrounding an interior having at least one aqueous portion, encapsulated inorganic particles, and an agent of interest (e.g., a therapeutic, diagnostic, or therapeutic diagnostic agent). The ionizable lipid is present at 2 mol% to 30 mol% of the total lipid. The agent of interest is a hydrophilic agent present in at least one aqueous portion or a lipophilic agent present in at least one lipid layer. The present application also relates to methods of making lipid nanoparticles and methods of using lipid nanoparticles (e.g., for therapy or diagnosis), which include using an external stimulus (e.g., laser irradiation) to release the agent of interest.

Description

[Technical field]

[0001] (Priority) This application claims priority to U.S. Provisional Patent Application No. 63 / 310,065, filed February 14, 2022, and U.S. Provisional Patent Application No. 63 / 340,678, filed May 11, 2022, each of which is incorporated by reference in its entirety.

[0002] (Technical field) The present disclosure relates to lipid nanoparticles (LNPs), particularly lipid nanoparticles comprising inorganic particles and a drug of interest, and methods for making the same. [Background technology]

[0003] Current therapies using small molecule therapeutic agents suffer from the limitation that less than 0.01% of a systemically administered drug is delivered to the target site, e.g., tumor tissue. Many efforts have been made to enhance delivery to the target tissue without harming sensitive organs. Examples include implantable (macroscale) sustained release devices that slowly release drugs and systemically (intravenously) administered nanocarriers containing drug cargo that preferentially accumulate at disease sites, e.g., tumors. Implantable devices suffer from the need to be precisely positioned in the region of the tumor, among other limitations. Systemically administered nanocarriers, on the other hand, require stable drug encapsulation in order to be delivered to the disease site. However, stable encapsulation can prove challenging for drug release at the target site, as the encapsulated drug has limited bioavailability. Triggered release at the target site in response to an external stimulus could address this, but targeted release after reaching the disease site has proven difficult to achieve in practice.

[0004] Lipid nanoparticles (LNPs) are well-established nanocarriers for delivering a wide range of cargoes to target sites in the body (see, e.g., Bangham et al., 1965, J Mol Biol, vol. 13, no. 1, pp. 238-52; Allen and Cullis, 2013, Adv Drug Deliv Rev, vol. 65, no. 1, pp. 36-48; Brader et al., 2021, Biophysical Journal, vol. 120, no. 14, pp. 2766-2770). LNP drug delivery systems represent a mature technology for the delivery of small molecule drugs (e.g., anticancer drugs), with nine intravenously injectable LNP drugs approved by regulatory authorities worldwide. Various nanoparticles have been tested as potential delivery and imaging agents. Gold nanoparticles (GNPs or AuNPs) have been identified as promising imaging candidates (Jahangirian et al., Int J Nanomedicine 2019, 14, 1633-1657; and Fernandes et al., J Photochem Photobiol B 2021, 218, 112110) and have potential controlled release properties (Whitener et al., J Biomed Mater Res A, 2021, 109(7), 1256-1265). GNP systems have the additional potential to cause triggered release of contents, as they can be "exploded" in response to high-energy pulsed laser radiation (Letfullin, RR; Joenathan, C.; George, TF; Zharov, VP, Laser-induced explosion of gold nanoparticles: potential role for nanophotothermolysis of cancer. Nanomedicine (Lond) 2006, 1(4), 473-80). However, previously reported hybrid LNP-GNP systems typically lack one or more of the properties required for clinical utility.

[0005] There is a need in the art for nanoparticle formulations containing inorganic particles that offer improvements and / or useful alternatives to known formulations. Summary of the Invention

[0006] The embodiments disclosed herein represent improvements over, and / or provide a useful alternative to, previous efforts for entrapping both inorganic particles and therapeutic and / or imaging agents in lipid nanoparticles (LNPs).

[0007] In some embodiments, the present disclosure is based at least in part on the discovery that LNPs containing low levels of ionizable lipids exhibit unique morphology, such that both agents of interest (e.g., therapeutic agents or diagnostic / imaging agents) and inorganic particles are efficiently encapsulated in the same lipid nanoparticle. Specifically, such hybrid LNPs include an inner core with an aqueous portion that can be loaded with high levels of agents, while at the same time, such LNPs can accommodate high levels of inorganic particles therein. Lipid nanoparticles can be used for a wide range of clinical applications by triggered release of the contents of the LNP. Furthermore, such LNPs include lipid components and lamellar structures, which can enable long circulation lifetimes to reach extrahepatic tissues.

[0008] Various embodiments relate to lipid nanoparticles comprising a helper lipid and an ionizable lipid, where the ionizable lipid is present at 2 mol % to 30 mol % of the total lipid, at least one lipid layer surrounding an interior having at least one aqueous portion, encapsulated inorganic particles, and a drug of interest, where the drug of interest is a hydrophilic drug present in the at least one aqueous portion or a lipophilic drug present in the at least one lipid layer.

[0009] Further provided are methods for producing such LNPs that can facilitate high loading efficiency of therapeutic and / or imaging agents and that are scalable.

[0010] Various embodiments relate to a method for producing lipid nanoparticles having entrapped inorganic particles and an agent of interest, comprising: (i) combining a first preparation of lipids dissolved in a solvent and a second preparation of an aqueous solution of inorganic particles in two separate streams to produce a combined stream, thereby forming lipid nanoparticles in the combined stream having encapsulated inorganic particles; (ii) introducing a loading medium into an external solution of the lipid nanoparticles thereby formed, comprising a solvent, such that the loading medium is entrapped in the lipid nanoparticles, thereby producing lipid nanoparticles comprising inorganic particles and entrapped loading medium in their interior compartments; and (iii) introducing the agent of interest into the external solution of the lipid nanoparticles comprising the entrapped loading buffer, such that the lipid nanoparticles are actively loaded with the agent of interest in response to the entrapped loading medium, thereby producing lipid nanoparticles having entrapped inorganic particles and an agent of interest.

[0011] Various embodiments relate to a method for making lipid nanoparticles comprising an inorganic particle core and a lipophilic agent of interest, comprising combining a first preparation of lipids dissolved in a solvent and a second preparation of an aqueous solution of inorganic particles in two separate streams to produce a combined stream, thereby forming lipid nanoparticles in the combined stream encapsulating the inorganic particle core, wherein the lipids in the first preparation comprise a helper lipid, an ionizable lipid, and the lipophilic agent of interest, wherein the ionizable lipid is present at 2 mol% to 30 mol% of total lipid, optionally 5 mol% to 15 mol% of total lipid, and the helper lipid is present at a concentration of at least 20 mol%, optionally at least 30 mol%, optionally at least 40 mol%.

[0012] Various embodiments relate to the use of lipid nanoparticles as disclosed herein for delivering a desired agent to a subject, wherein the lipid nanoparticles are administered to the subject and subsequently a stimulus is administered to an area of ​​the subject, the stimulus causing the inorganic particles in the lipid nanoparticles to release the desired agent from the lipid nanoparticles.

[0013] In some embodiments, the present disclosure relates to lipid nanoparticles composed of metal nanoparticles. In a particular example, the lipid nanoparticles contain two separate internal layers / chambers (e.g., bilayers) for encapsulating drugs in the aqueous core and metal nanoparticles in the lipid layer, respectively. Utilizing pulsed femtosecond and nanosecond lasers to provide a local energy distribution of a few nanoseconds within a few nanometers, the formulated liposomes / plasmonic nanocarriers are used for site-specific light-triggered delivery of drugs (e.g., pH-gradient-loadable drugs, e.g., Dox) into cells in vitro or in vivo.

[0014] Various embodiments relate to lipid nanoparticles comprising an ionizable lipid content of 2 mol% to 30 mol%, at least one of a hydrophilic polymer-lipid conjugate and a sterol, a helper lipid content of greater than 30 mol% that forms a bilayer surrounding an aqueous portion, inorganic particles present in the bilayer, and a therapeutic and / or imaging agent present in the aqueous portion, wherein the therapeutic and / or imaging agent is releasable from the lipid nanoparticles by irradiation.

[0015] Various embodiments relate to the use of the lipid nanoparticles disclosed herein to treat a subject (e.g., a mammalian subject), including triggered release of an agent at a bodily target site. Various embodiments relate to a method of medical treatment comprising administering the lipid nanoparticles disclosed herein to a subject (e.g., a mammalian subject) in need of such treatment, and subjecting the lipid nanoparticles to irradiation to trigger release of the agent at the bodily target site.

[0016] Other objects, features and advantages of the present disclosure will become apparent to those skilled in the art from the following detailed description and drawings. [Brief description of the drawings]

[0017] [Figure 1A]Figure 1A shows the encapsulation of gold nanoparticles in a hybrid LNP system with the lipid composition DODAP / DSPC / Chol / PEG-DSPE, 10 / 49 / 40 / 1 mol / mol) visualized by cryo-TEM. Figure 1A shows LNPs formed in the absence of GNPs at pH 4. Size bar represents 100 nm. [Figure 1B] Figure 1B shows the encapsulation of gold nanoparticles in a hybrid LNP system with the lipid composition DODAP / DSPC / Chol / PEG-DSPE, 10 / 49 / 40 / 1 mol / mol) visualized by cryo-TEM. Figure 1B shows LNPs formed in the absence of GNPs at pH 4 and then dialyzed against PBS. Size bar represents 100 nm. [Figure 1C] Figure 1C shows the encapsulation of gold nanoparticles in a hybrid LNP system with lipid composition DODAP / DSPC / Chol / PEG-DSPE, 10 / 49 / 40 / 1 mol / mol) visualized by cryo-TEM. Figure 1C shows LNP-GNPs formed at pH 4, Au / L 2.2 x 1013 particles / μmol lipid. Arrows indicate "dumbbells" where GNPs bridge two vesicles. Size bar represents 100 nm. [Figure 1D] Figure ID shows encapsulation of gold nanoparticles in hybrid LNP systems with lipid composition DODAP / DSPC / Chol / PEG-DSPE, 10 / 49 / 40 / 1 mol / mol) visualized by cryo-TEM. Figure ID shows LNP-GNPs formed at pH 7.4, Au / L 2.2 x 1013 particles / μmol lipid. Size bars represent 100 nm. [Figure 1E] Figure 1E shows the encapsulation of gold nanoparticles in a hybrid LNP system with lipid composition DODAP / DSPC / Chol / PEG-DSPE, 10 / 49 / 40 / 1 mol / mol) visualized by cryo-TEM. Figure 1E shows LNP-GNP formed at pH 7.4, Au / L 6.6×1013 particles / μmol lipid. Size bar represents 100 nm. [Figure 1F]Figure IF shows the encapsulation of gold nanoparticles in a hybrid LNP system with lipid composition DODAP / DSPC / Chol / PEG-DSPE, 10 / 49 / 40 / 1 mol / mol) visualized by cryo-TEM. Figure IF shows LNP-GNP formed at pH 7.4, Au / L 8.8 x 1013 particles / μmol lipid. Size bar represents 100 nm.

[0018] [Diagram 2] Normalized absorbance (au) at 520 nm versus initial AuNP / lipid ratio for LNP-GNP formulations with lipid composition DODAP / DSPC / Chol / PEG-DSPE, 10 / 49 / 40 / 1 mol / mol, measured after removal of untrapped gold (lipid concentration normalized to 1.25 mg / ml). Inset shows normalized absorbance spectra obtained from LNP-GNP systems after removal of untrapped gold (Au / L ratios, from top to bottom: 8.8×1013, 6.6×1013, 4.4×1013, 3.3×1013, 2.2×1013, 1.1×1013 / μmol lipid).

[0019] [Diagram 3] 1 is a bar graph showing the % AuNP capture as a function of the initial AuNP / lipid ratio for the ratios of LNP-GNP formulations with lipid composition DODAP / DSPC / Chol / PEG-DSPE, 10 / 49 / 40 / 1 mol / mol. The Au / L ratios used were: (1) 1.1×1013 / μmol lipid, (2) 2.2×1013 / μmol lipid, (3) 3.3×1013 / μmol lipid, (4) 4.4×1013 / μmol lipid, (5) 6.6×1013 / μmol lipid, (6) 8.8×1013 / μmol lipid.

[0020] [Figure 4A]FIG. 4A is a bar graph showing entrapment of ammonium sulfate (AS) in LNP-GNP formulations (DODAP / DSPC / Chol / PEG-DSPE, 10 / 49 / 40 / 1 mol / mol) prepared containing GNPs (Au / L ratio 2.2×1013 / μmol) and loaded with AS by adding AS after LNP formation at pH 4, followed by dialysis against PBS to remove ethanol and increase the pH to pH 7.4. The AS concentrations in the pH 4 formulation mixtures were as follows: (1) 300 mM, (2) 450 mM, (3) 600 mM. FIG. 4A shows the entrapped ammonium to lipid ratio (mol / mol). [Figure 4B] FIG. 4B is a bar graph showing entrapment of ammonium sulfate (AS) into LNP-GNP formulations (DODAP / DSPC / Chol / PEG-DSPE, 10 / 49 / 40 / 1 mol / mol) prepared containing GNPs (Au / L ratio 2.2×1013 / μmol) and loaded with AS by adding AS after LNP formation at pH 4, followed by dialysis against PBS to remove ethanol and increase the pH to pH 7.4. The AS concentrations in the pH 4 formulation mixtures were as follows: (1) 300 mM, (2) 450 mM, (3) 600 mM. FIG. 4B shows the corresponding ammonium entrapment efficiency after dialysis against PBS.

[0021] [Figure 5A] Figure 5A shows the doxorubicin entrapment (%) versus time for ammonium sulfate-loaded LNP-GNP formulations. Figure 5A shows doxorubicin loading into LNP-GNP systems prepared in the presence of 300 mM (■), 450 mM (●), and 600 mM (▲) AS at the pH 4 stage of the formulation and subsequently dialyzed against PBS (initial drug-to-lipid ratio of 0.1 wt / wt). [Figure 5B] Figure 5B shows the doxorubicin entrapment (%) versus time for ammonium sulfate-loaded LNP-GNP formulations. Figure 5B shows the doxorubicin entrapment at various drug-to-lipid ratios (0.05 wt / wt (■), 0.1 wt / wt (●), and 0.2 wt / wt (▲)) using LNP-GNP systems prepared in 450 mM AS at pH 4 step.

[0022] [Figure 6A] Figure 6A shows the design and properties of an exemplary LNP / AuNP / Dox system. Figure 6A is a cryo-TEM image of LNPs (DODAP / DSPC / Chol / PEG-DSPE, 10 / 49 / 40 / 1 mol / mol) containing AuNPs (Au / L 2.2x1013 / μmol) prepared in 450 mM ammonium sulfate (AS) and loaded with doxorubicin at a drug-to-lipid ratio of 0.1 (wt / wt). The drug forms rod-shaped precipitates in the center of the LNP-AuNPs, where the black arrow points. The black dots are 5 nm AuNPs. The LNPs are spherical with an average diameter of 100 nm. [Figure 6B] Figure 6B shows the design and properties of an exemplary LNP / AuNP / Dox system. Figure 6B is a cryo-TEM image of the LNP / AuNP system. The morphology of the LNPs is similar to that with Dox in the aqueous core. [Figure 6C] Figure 6C shows the design and properties of an exemplary LNP / AuNP / Dox system. Figure 6C is a schematic diagram of various LNP samples. Dox is in the aqueous core and gold nanoparticles are located in the outer layer of the LNP. [Figure 6D] Figure 6D shows the design and properties of an exemplary LNP / AuNP / Dox system. Figure 6D is a table showing the physicochemical properties of the LNPs, namely, the hydrodynamic diameter, size distribution (PDI), and zeta potential of control LNPs and LNPs with and without AuNPs and Dox (measured at the same concentration of Dox of 0.1 mg / mL). [Figure 6E] Figure 6E shows the design and properties of an exemplary LNP / AuNP / Dox system. Figure 6E shows the fluorescence spectra of LNP / AuNP / Dox (lower spectrum) and LNP / Dox (upper spectrum) from 570 nm to 610 nm using excitation from 505 to 555 nm. The fluorescence is due to Dox, which is naturally fluorescent. [Figure 6F]Figure 6F shows the design and properties of an exemplary LNP / AuNP / Dox system. Figure 6F shows the absorbance spectra of LNP / AuNP / Dox (maximum at the top), LNP / Dox (maximum at the second top), LNP / AuNP (maximum at the second bottom), LNP (maximum at the bottom), and Dox (maximum in the middle). Spectra were measured with the same concentration of Dox or the corresponding concentration of LNP.

[0023] [Figure 7A] FIG. 7A shows the release of Dox after irradiation with a nanosecond laser with three different protocols. FIG. 7A shows fluorescence images of MDA-MB-231 cells treated with control LNPs (without AuNPs) and with AuNPs, with and without irradiation. Concentration of LNPs at 50 μg / mL. Cell membrane staining with Calcein AM in green fluorescence and Dox-associated red fluorescence. Irradiation was performed with a numerical aperture NA=0.13 using a 4x objective, with an energy of 14.2 μJ, a scanning speed of 50 μm / s, and scan lines separated by a step of 5 μm. The pulse duration is 7.7 ns. The protocol for performing these irradiations is diagrammed below the images. Irradiation was performed before the washing step, which took place 4 hours after the addition of nanoparticles to the cells. The imaging step took place after 20 hours. For the control samples (i.e. without irradiation but with the same incubation time, the same waiting time before the photography and washing steps), the exact same protocol was followed. [Figure 7B] Figure 7B shows the release of Dox after nanosecond laser irradiation with three different protocols. Figure 7B shows fluorescence images of MDA-MB-231 cells incubated with LNP / AuNP / Dox for 1 h at a concentration of 50 μg / mL of LNP. Irradiation was performed as follows: image 1 after the washing step, image 2 just before the washing step, and image 3 without irradiation. Irradiation was performed with an energy of 19.7 μJ, 50 μm / s, and 5 μm steps. The exact protocol followed is detailed below the three images. Two irradiations were performed for two different areas, one before and one after the washing step. [Figure 7C]FIG. 7C shows the release of Dox after irradiation with a nanosecond laser with three different protocols. FIG. 7C shows fluorescence images of MDA-MB-231 cells treated with control LNPs (without AuNPs) and with AuNPs, with and without irradiation. Concentration of LNPs of 50 μg / mL. Irradiation was performed with an energy of 19.7 μJ, 50 μm / s, and a step of 5 μm. Calcein AM staining of the cell membrane in green fluorescence and Dox-associated red fluorescence. The protocol schematized below the images corresponds to irradiation performed after cell washing. LNPs were in solution with the cells for only 15 min.

[0024] [Figure 8] Figure 1 shows the normalized fluorescence intensity of LNPs containing Dox with or without gold nanoparticles (AuNPs) after irradiation with a nanosecond laser at different energies (0-19.7 μJ) with two different scanning parameters: speed 1: 100 μm / s and 10 μm steps, and speed 2: 50 μm / s and 5 μm steps. The normalized values ​​were fixed to 1 for the non-irradiated samples of LNP / Dox. Cell incubation was performed for 4 h with LNP / Dox and LNP / AuNP / Dox at a concentration of 50 μg / mL.

[0025] [Figure 9A] Figure 9A shows the release of Dox after irradiation with a femtosecond laser (wavelength 800 nm, pulse width 55 fs, repetition rate 1 kHz, spot diameter 35 μm). Figure 9B shows fluorescence images of MDA-MB-231 cells treated with control LNPs (no AuNPs) and with AuNPs, with and without irradiation. The irradiation protocol is diagrammed below the images. Cell incubations used a concentration of 50 μg / mL LNP / Dox or LNP / AuNP / Dox. [Figure 9B]Figure 9B shows the release of Dox after irradiation with a femtosecond laser (wavelength 800 nm, pulse width 55 fs, repetition rate 1 kHz, spot diameter 35 μm). Figure 9B shows the normalized fluorescence intensity of LNPs containing Dox after irradiation with a femtosecond laser at different fluences (0-73 mJ / cm2) with two different scanning parameters, namely, speed 1: 3 cm / s and step of 30 μm, and speed 2: 6 cm / s and step of 60 μm. The normalized value was fixed to 1 for the non-irradiated sample of LNP / Dox. [Figure 9C] Figure 9C shows the release of Dox after irradiation with a femtosecond laser (wavelength 800 nm, pulse width 55 fs, repetition rate 1 kHz, spot diameter 35 μm). Figure 9C shows a schematic diagram of the irradiation space for each speed. Each dot represents a laser pulse and each arrow represents the laser path. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] <Lipid nanoparticles>

[0027] The lipid nanoparticles (LNPs) described herein comprise helper lipids and cationic lipids at levels selected to produce LNPs with morphology particularly suitable for efficient encapsulation of both inorganic particles (e.g., metal nanoparticles) and agents (e.g., therapeutic or diagnostic agents), which agents can be loaded, for example, by active loading methods. The helper lipids can be present at greater than 30 mol%. In another embodiment, the cationic lipids are present at 2 mol% to 30 mol% of the total lipid. Such LNPs are particularly well suited for triggered release of the contents of the LNP in therapeutic or diagnostic applications.

[0028] In one non-limiting example, a lipid layer, e.g., a bilayer or other lamella structure, surrounds the interior of the LNP, which includes an aqueous portion. As used herein, the "interior" of the LNP (also referred to as the "inner core" or simply "core") refers to everything inside the outermost lipid layer (e.g., the outermost bilayer) that separates the lipid nanoparticle from its external environment. It has been observed that as the percentage of ionizable lipids is further reduced, the size of the hydrophobic core may decrease and the number of lamellae decreases. Without limitation, an LNP incorporating inorganic particles as described herein, e.g., at their maximum encapsulation levels, may essentially be free of a hydrophobic core within the inner core or a small region thereof. In such embodiments, the inorganic particles may be at least partially complexed with ionizable lipids. Without limitation, the inorganic particles may be located at the intersection of one or more lipid layers (e.g., lamellae). Advantageously, the aqueous portion in such lipid nanoparticles may accommodate actively loaded therapeutic and / or diagnostic agents with high encapsulation efficiency.

[0029] Lipid nanoparticles typically have an average diameter of 50-180 nm, 60-150 nm, or 65-130 nm, or any range therebetween. Lipid nanoparticles may be elongated or circular in cross-section.

[0030] <Helper lipids>

[0031] In the context of this disclosure, the term "helper lipid" includes any vesicle-forming or liposome-forming lipid. Thus, helper lipids include amphipathic lipids (e.g., C14-C18 alkyl chains with 0-3 double bonds) whose polar (i.e., hydrophilic) region contains a phosphate, carboxyl, sulfate, sulfonyl, amino, or nitro group. In some embodiments, the helper lipid is a phospholipid (e.g., phosphatidylcholine, sphingomyelin, etc., or mixtures thereof). The helper lipid may be cationic, anionic, or zwitterionic at physiological pH (e.g., pH about 7.0) and may have a net negative charge, a net positive charge, or a net neutral charge. In some embodiments, the helper lipid has a net neutral charge. Non-limiting examples of helper lipids include DLPC(12:0), DMPC(14:0), DPPC(16:0), DSPC(18:0), DOPC(18:1), DMPE(14:0), DPPE(16:0), DOPE(18:1), DMPA(14:0), DPPA(16:0), DOPA(18:1), DMPG(14:0), DPPG(16:0), DOPG(18:1), DMPS(14:0), DPPS(18:1), DOPS(18:1), DPOE-glutaryl(14:0), tetramyristoylcardiolipin(14:0), DOTAP(18:1), and in some embodiments, the helper lipid is one or a mixture of two or more of the foregoing. In some embodiments, the helper lipid is DLPC(12:0), DMPC(14:0), DPPC(16:0), DSPC(18:0), DOPC(18:1), DMPE(14:0), DPPE(16:0), DOPE(18:1), or a combination of two or more thereof. In some embodiments, the helper lipid is selected from sphingomyelin, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), dipalmitoyl-phosphatidylcholine (DPPC), or a mixture of two or more thereof.In certain embodiments, the helper lipid is DOPC, DSPC, sphingomyelin, or a mixture of two or more thereof. In one embodiment, the helper lipid is DSPC. The helper lipid content can be one helper lipid or a mixture of two or more different helper lipids.

[0032] In some embodiments, the helper lipid content is more than 20 mol%, more than 25 mol%, more than 30 mol%, more than 32 mol%, more than 34 mol%, more than 36 mol%, more than 38 mol%, more than 40 mol%, more than 42 mol%, more than 44 mol%, more than 46 mol%, more than 48 mol%, or more than 50 mol%. In some embodiments, the upper limit of the helper lipid content is 70 mol%, 65 mol%, 60 mol%, 55 mol%, 50 mol%, or 45 mol%. The present disclosure also encompasses subranges that combine any combination of the upper and lower limits of the aforementioned numerical values.

[0033] For example, in certain embodiments, the total helper lipid content is between 20 mol% and 70 mol%, or between 25 mol% and 70 mol%, or between 30 mol% and 70 mol%, or between 35 mol% and 70 mol%, or between 40 mol% and 70 mol% of the total lipid present in the lipid nanoparticle.

[0034] In some embodiments, the phosphatidylcholine content of the lipid nanoparticles is greater than 15 mol%, greater than 20 mol%, greater than 25 mol%, greater than 30 mol%, greater than 32 mol%, greater than 34 mol%, greater than 36 mol%, greater than 38 mol%, greater than 40 mol%, greater than 42 mol%, greater than 44 mol%, greater than 46 mol%, greater than 48 mol%, or greater than 50 mol%. In some embodiments, the upper limit of the phosphatidylcholine content is 70 mol%, 65 mol%, 60 mol%, 55 mol%, 50 mol%, or 45 mol%. The present disclosure also encompasses subranges that combine any of the upper and lower limits of the aforementioned numerical values. For example, in certain embodiments, the phosphatidylcholine content is 20 mol% to 60 mol%, or 25 mol% to 60 mol%, or 30 mol% to 60 mol%, or 35 mol% to 60 mol%, or 40 mol% to 60 mol% of the total lipid present in the lipid nanoparticle. The phosphatidylcholine lipid content is determined based on the total amount of lipid, including sterols, in the lipid nanoparticle.

[0035] <Ionizable lipids>

[0036] The term "ionizable lipid" refers to any of several lipid species that have a net positive or negative charge at a selected pH, such as physiological pH (e.g., a pH of about 7.0). Ionizable lipids can be cationic, anionic, or zwitterionic.

[0037] In some embodiments, one or more ionizable lipids comprise cationic lipids, which in certain embodiments have a head group comprising an amino group. In selected embodiments, the cationic lipid comprises a protonatable tertiary amine (e.g., pH-titratable) head group, a C14-C18 alkyl chain, a linker region between the head group and the alkyl chain, and 0-3 double bonds. Non-limiting examples of cationic ionizable lipids include 1,2-dioleoyl-3-dimethylammonium propane (DODAP), 1,2-dioleyloxy-3-dimethylaminopropane (DODMA). Such lipids include, but are not limited to, DLin-KC2-CMA (KC2), DLin-MC3-DMA (MC3), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), 1,2-dioleyloxy-3-dimethylaminopropane (DODMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleoyl-3-(trimethylammonium)propane (DOTAP), and 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA). In some embodiments, the ionizable lipid is one or a mixture of two or more cationic lipids, such as two or more of those disclosed herein.

[0038] In some embodiments, the one or more ionizable lipids comprise an anionic lipid. In selected embodiments, the anionic lipid comprises an anionic head group (e.g., phosphate, carboxyl, sulfate, sulfonyl, nitro, etc.), a C14-C18 alkyl chain, a linker region between the head group and the alkyl chain, and 0-3 double bonds, and is optionally pegylated (PEG attached to the head group). Non-limiting examples of anionic ionizable lipids include DMPA (14:0), DPPA (16:0), DOPA (18:1), DMPG (14:0), DPPG (16:0), DOPG (18:1), DMPS (14:0), DPPS (18:1), DOPS (18:1), DPOE-glutaryl (14:0), tetramyristoyl cardiolipin (14:0). In certain embodiments, a mixture of ionizable lipids is included in the lipid nanoparticle.

[0039] In some embodiments, the ionizable lipid comprises one or more charged lipids as described in WO 2021 / 026647, which is incorporated by reference in its entirety. In some such embodiments, the one or more charged lipids are one or more lipids comprising a branched lipid moiety L having the structure of Formula I (term definitions incorporated by reference from WO 2021 / 026647):

[0040] Formula I: A-(V) m -ZL (I) A is a head group that is ionizable, permanently charged, or zwitterionic; (V) m is any -(CR1R2) m -, m is 1 to 10 or 2 to 6, and R1 and R2 are each independently hydrogen, optionally substituted alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkylalkyl, or heterocycle; ZL has the structure of formula II, IIa, or IIb: L is a hydrocarbon structure having the moiety of formula IIIc:

[0041] Formula IIIc: [ka] In the formula, the scaffold carbon chain of L is L1'-L1''-G 1 -CH-[CH2] q The total number of carbon atoms in the L carbon skeleton is 10 to 30; L1' is a linear hydrocarbon chain having no heteroatoms and having 5 to 12 (or more) carbon atoms and 0 to 3 cis or trans double bonds; L1″ is a carbon atom; each X1 is independently selected from an ether, ester, and carbamate group; L1''' is the carbon skeleton of the scaffold carbon chain L, and G 1 -CH-[CH2] q In the formula, G is represented by —CH 1 is a hydrocarbon chain of 0 to 4 carbon atoms, optionally containing one cis or trans double bond; n is 0 to 4; p is 1 to 4; n+p is 1 to 4, q is 0 to 20; Each S and L1'''' is a hydrocarbon side chain and is independently (a) a linear or branched terminal hydrocarbon chain having 0 to 5 cis or trans C=C and 2 to 30 carbon atoms, conjugated to one of the X1 at any carbon atom in the hydrocarbon chain; or (b) a branched hydrocarbon structure of formula IIIc, Each of the hydrocarbon chains of L1'''' and S in the lipid moiety is optionally substituted with heteroatoms, provided that no more than two heteroatoms are substituted in the hydrocarbon chain.

[0042] Formula II Linear Linker Structure: X1-L b , wherein X1 is optionally present and is selected from ether, ester, and carbamate groups; L b is a branched lipid of formula IIIc.

[0043] Formula IIa Branched Linker Structure: [ka] W is optionally present; W, if present, is an X bond, NC(O), NC(O)O, or N-OC(O); W is optionally substituted with D, where D is an optionally substituted alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkylalkyl, or heterocycle; (X) n Each occurrence of is selected independently - (CR1R2) n - and (X) n n is 0-10; T is optionally present and is alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkylalkyl, or heterocycle; T is optionally substituted; B is a carbon atom bonded to L1 and L2 via G1 and G2, respectively; G1 and G2 are independently selected from X1; Each of G1 and G2 is independently an intervening (G) u through the group B-(G) u -G1 or B-(G) u - optionally covalently bonded to B as G2, (G) u are independently selected -(CR1R2) u wherein R1 and R2 are each independently hydrogen, optionally substituted alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkylalkyl, or heterocycle; and u is 0 to 16; G3 is optionally present and selected from X1 and includes an intervening (G) u Through the group, B-(G) u - optionally covalently linked to B as G3; L1 is a branched hydrocarbon of formula IIIc, L2 is a hydrocarbon chain having 1 to 20 carbon atoms and 0 to 2 cis or trans double bonds or has the structure of formula IIIc, L3, when present, is hydrogen, a straight or branched hydrocarbon chain having 1 to 20 carbon atoms and 0 to 2 cis or trans double bonds, or has the structure of formula IIIc.

[0044] Formula IIb Ring structure: [ka] In the formula, the curved line represents a ring, E and K represent atoms partially forming the ring structure, and the ring is a substituted or unsubstituted ring having 3 to 8 ring atoms; At least one of L1, L2, and L3 is optionally bonded to an atom in the ring via G1, G2, and G3, respectively, and each of G1, G2, and G3 is independently an intermediate (G) u through G1-(G), respectively. u -L1, G2--(G) u -L2 or G3-(G) u -L3 is optionally covalently linked to each one of L1, L2, and L3; L1, and optionally L2 and / or L3, of formula IIb have the structure of formula IIIc, The A moiety is optionally selected from any of the following: (i) an ionizable cationic moiety selected from the group consisting of: [ka] , (ii) a permanently charged moiety selected from the group consisting of: [ka] (iii) an ionizable anionic moiety selected from the group consisting of: [ka] or (iv) a zwitterionic moiety selected from the group consisting of: [ka]

[0045] In some embodiments, ZL has the structure of formula II, L1' of formula IIIc has 5-9 carbon atoms and 0-2 cis or trans double bonds, and G 1 is absent, is CH2 or CH2CH=CH, the double bond is cis or trans, L1'''' if present in formula IIIc, and S are independently selected from 0-5 cis or trans CH=CH and a hydrocarbon having 2-18 carbon atoms, and q is 1-9.

[0046] In some embodiments, (V) m (CH2) m wherein m is 1 to 20; ZL has the structure of formula IIa, wherein W is an ether group, an ester group, or a carbamate group; D is absent; (X) n (CH2) n wherein n is 1 to 10, G1 and G2 are present, and (G) u Via B-(G) u -L1 or B-(G) u -L2 is covalently attached to B, wherein: u (CH2) uand G3-L3 is present and is a hydrocarbon selected from CH3 and CH2CH3, or G3-L3 is CH2X1L3 and L3 is a straight or branched hydrocarbon chain having 1 to 20 carbon atoms and 0 to 2 cis or trans double bonds, or has the structure of formula IIIc.

[0047] In some embodiments, ZL has the structure of formula IIb, where the curved line represents a ring, E and K represent atoms partially forming the ring structure, and the ring is a substituted or unsubstituted carbocyclic ring having 3 to 6 ring atoms. In selected embodiments, the ring contains 3 or 5 carbon atoms. In selected embodiments, at least L1 and L2 are present and are attached to the ring via G1 and G2 groups, respectively, and each G1 and G2 group is connected to an intermediate (G) u is optionally covalently bonded to a ring atom via (G) u (CH2) u and u is an integer of 0 to 10 or 0 to 6.

[0048] In some embodiments, (V) m R1 or R2 is cycloalkyl, which is an optionally substituted monocyclic, bicyclic, or tricyclic carbocyclic ring.

[0049] In some embodiments, (V) m R1 and R2 are each independently selected from heteroatom rings having 4 to 12 ring atoms.

[0050] The ionizable lipid content can be less than 30 mol%, less than 25 mol%, less than 20 mol%, less than 18 mol%, less than 15 mol%, less than 12 mol%, less than 10 mol%, or less than 5 mol%. In certain embodiments, the ionizable lipid content is between 2 mol% and 30 mol%, or between 5 mol% and 25 mol%, or between 7 mol% and 20 mol% of the total lipid present in the lipid nanoparticle. In some embodiments, the ionizable lipid is cationic at physiological pH. In one embodiment, the amine to phosphate charge ratio (N / P) of the lipid nanoparticle is between 3 and 15, between 5 and 10, between 6 and 9, between 8 and 10, or between 5 and 8.

[0051] <Sterol>

[0052] The lipid nanoparticles may optionally contain a sterol. Examples of sterols include cholesterol or cholesterol derivatives such as cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, beta-sitosterol, fucosterol, etc. In one embodiment, the sterol is present at 15 mol% to 65 mol%, 18 mol% to 50 mol%, 20 mol% to 50 mol%, 25 mol% to 50 mol%, or 30 mol% to 50 mol%, based on the total lipids present in the lipid nanoparticles. In another embodiment, the sterol is cholesterol and is present at 15 mol% to 65 mol%, 18 mol% to 50 mol%, 20 mol% to 50 mol%, 25 mol% to 50 mol%, or 30 mol% to 50 mol%, based on the total lipids and sterols present in the lipid nanoparticles. In one embodiment, the sum of (i) the sterol content (e.g., cholesterol or a cholesterol derivative thereof) and (ii) the helper phospholipid content (e.g., phosphatidylcholine or sphingomyelin) is at least 50 mol%, at least 55 mol%, at least 60 mol%, at least 65 mol%, at least 70 mol%, at least 75 mol%, at least 80 mol%, or at least 85 mol%, based on the total lipid present in the lipid nanoparticle.

[0053] <Hydrophilic polymer-lipid conjugate>

[0054] In one embodiment, the lipid nanoparticle comprises a hydrophilic polymer-lipid conjugate that can be incorporated into the lipid nanoparticle. The conjugate comprises a vesicle-forming lipid having a polar head group and (ii) a hydrophilic polymer chain covalently attached to the head group. Examples of hydrophilic polymers include polyethylene glycol (PEG) (Nunes et al., 2019, Drug Deliv Transl Res, vol. 9, no. 1, pp. 123-130), polyvinylpyrrolidone, polyvinylmethylether, polyhydroxypropylmethacrylate, polyhydroxypropylmethacrylamide, polyhydroxyethylacrylate, polymethacrylamide, polydimethylacrylamide, polymethyloxazoline, polyethyloxazoline, polyhydroxyethyloxazoline, polyhydroxypropyloxazoline, polysarcosine, and polyaspartamide. In one embodiment, the hydrophilic polymer-lipid conjugate is a PEG-lipid conjugate. Non-limiting examples of PEG-lipid conjugates include DMPE-mPEG-2000(14:0), DMPE-mPEG-5000(14:0), DSPE-mPEG-2000(18:0), DSPE-mPEG-5000(18:0), DSPE-maleimide PEG-2000(18:0), DMG-PEG-2000(14:0), DSG-PEG-2000(18:0), and the like.

[0055] The hydrophilic polymer-lipid conjugate may be present in the nanoparticles at 0 mol% to 5 mol%, or 0.5 mol% to 3 mol%, or 0.5 mol% to 2.5 mol%, or 0.5 mol% to 2.0 mol%, or 0.5 mol% to 1.8 mol% of the total lipid. In another embodiment, the PEG-lipid conjugate is present in the nanoparticles at 0 mol% to 5 mol%, or 0.5 mol% to 3 mol%, or 0.5 mol% to 2.5 mol%, or 0.5 mol% to 2.0 mol%, or 0.5 mol% to 1.8 mol% of the total lipid. In certain embodiments, the PEG-lipid conjugate may be present in the nanoparticles at 0 mol% to 5 mol%, or 0 mol% to 3 mol%, or 0 mol% to 2.5 mol%, or 0 mol% to 2.0 mol%, or 0 mol% to 1.8 mol% of the total lipid.

[0056] <Inorganic particles>

[0057] The term "inorganic particles" refers to nano-sized particles suitable for formulation into lipid nanoparticles as described herein and containing a suitable metal. The inorganic particles have suitable properties to trigger drug release when exposed to a suitable energy wavelength. For example, the inorganic particles may be magnetic. The metals in some non-limiting examples are most advantageously biocompatible and non-toxic. Metals include, but are not limited to, gold, silver, iron, copper, nickel, cobalt, platinum, iridium, alloys of two or more thereof, or mixtures thereof. The metals may be in any form, such as a salt (e.g., oxide, hydroxide, sulfide, phosphate, fluoride, or chloride), or may be complexed. In some embodiments, the metal is gold or silver. In another embodiment, the inorganic particles include hybrid gold-iron oxide.

[0058] Inorganic particles include, but are not limited to, metal nanoparticles, nanoshells, nanocages, quantum dots, or upconverting nanoparticles. In some embodiments, inorganic particles are in the shape of a sphere or rod or nanostar, most typically a sphere. Inorganic particles are typically small, with a diameter of less than 20 nm, less than 15 nm, less than 10 nm, or less than 5 nm.

[0059] In the case of metal nanoparticles, the metal may be associated with a ligand, e.g., a "capping agent." Without limitation, the capping agent may control the growth, aggregation, and / or physicochemical properties of the metal nanoparticles. The capping agent, in some embodiments, may reduce or block reactivity at the periphery of the metal nanoparticles. The capping agent, in some embodiments, may function as both a reducing agent and a capping agent.

[0060] In certain advantageous embodiments, the capping agent imparts a negative charge to the metal nanoparticles. If the ionizable lipid is cationic at the desired pH (e.g., at or below physiological pH), the use of a negatively charged capping ligand can facilitate the incorporation of the metal nanoparticle into the lipid nanoparticle. In some embodiments, the opposite charge between the ionizable lipid and the metal nanoparticle allows the formation of an association or complex between the negatively charged metal nanoparticle and the positively charged lipid, thereby improving the encapsulation efficiency. In one embodiment, the capping agent is a macromolecule, such as, but not limited to, tannic acid or includes citrate ions.

[0061] The capping agent, in some alternative embodiments, can be an amphiphilic molecule that includes a polar head group and a non-polar hydrocarbon tail. Due to its amphiphilic nature, the capping agent, in some embodiments, provides functionality and / or enhances affinity with another phase. In one non-limiting example, the non-polar tail interacts with the external medium and the polar head interacts with the metal atom of the nanoparticle.

[0062] <Filling the desired drug>

[0063] In some embodiments, the LNPs comprise one or more aqueous soluble loadable agents of interest present in the aqueous portion of the LNP, e.g., one or more therapeutic, diagnostic, and / or therapeutic diagnostic agents. In other embodiments, the LNPs comprise one or more hydrophobic or lipophilic loadable agents of interest present in the lipid portion of the LNP, e.g., one or more therapeutic, diagnostic, and / or therapeutic diagnostic agents, or prodrugs thereof. The loadable agent of interest can be any molecule of interest, e.g., a small molecule (e.g., a small molecule drug, imaging agent, etc.), a protein (e.g., an antibody, etc.), a peptide, a nucleic acid (e.g., siRNA, etc.).

[0064] In one embodiment, the drug incorporated into the aqueous portion of the lipid nanoparticle is capable of active loading into the lipid nanoparticle. Drugs that can be loaded using pH gradient loading include one or more ionizable moieties, so that the neutral form of the ionizable moiety allows the drug to pass through the lipid nanoparticle membrane, and when the ionizable moiety is converted to a charged form, the drug remains encapsulated within the liposome. The ionizable moiety may include an amine, a carboxylic acid, a hydroxyl group, or some other charged moiety. Drugs that load in response to an acidic interior may include an ionizable moiety that charges in response to an acidic environment, while drugs that load in response to a basic interior include a moiety that charges in response to a basic environment. In the case of a basic interior, ionizable moieties including, but not limited to, carboxylic acid or hydroxyl groups may be utilized. In the case of an acidic interior, ionizable moieties including, but not limited to, primary, secondary, and tertiary amine groups may be used. In some embodiments, drugs loaded into a basic interior using pH gradient loading should be weak acids or have a pKa of about 2-6 and a molecular weight <1500 g / mol, while drugs loaded into an acidic interior using pH gradient loading should be weak bases or have a pKa of about 6-9 and a molecular weight <1500 g / mol.

[0065] Without intending to be limiting, the pH gradient loadable drug can be an anti-neoplastic agent, an anti-bacterial agent, or an anti-viral agent. Non-limiting examples of therapeutic agents that can be loaded into lipid nanoparticles by the pH gradient loading method and therefore can be used in the practice of the present disclosure include, but are not limited to, anthracycline antibiotics such as doxorubicin, daunorubicin, mitoxantrone, epirubicin, aclarubicin, and idarubicin; antineoplastic antibiotics such as mitomycin, bleomycin, and dactinomycin; vinca alkaloids such as vinblastine, vincristine, and navelbine; purine derivatives such as 6-mercaptopurine and 6-thioguanine; purine and pyrimidine derivatives such as 5-fluorouracil; camptothecins such as topotecan, irinotecan, rutotecan, 9-aminocamptothecin, 9-nitrocamptothecin, and 10-hydroxycamptothecin; cytarabine such as cytosine arabinoside; antibacterial agents such as ciprofloxacin and its salts.

[0066] In some embodiments, the loadable agent of interest is a diagnostic or imaging agent (e.g., a contrast agent, e.g., a radiolabeled or MRI contrast agent, a fluorescent probe, etc.). Such agents may be incorporated into the aqueous portion of the lipid nanoparticle or into the lipid portion of the lipid nanoparticle. For example, but not limited to, one or more soluble contrast agents may be incorporated into the aqueous portion of the LNP.

[0067] Hydrophobic drugs of interest can be easily loaded into the LNP system by simply mixing with the lipid components (see, eg, Example 4).

[0068] Hydrophilic drugs can be converted to hydrophobic drugs using known methods, such as those described in WO 2020 / 191477, which is incorporated by reference in its entirety, and thus can be lipid-loaded, including but not limited to conjugating lipid moieties. The lipid moieties can be conjugated using various linkers, such as succinates, esters, amides, hydrazones, ethers, carbamates, carbonates, phosphodiesters, and the like. The lipid-conjugated drug can be a therapeutic agent, a diagnostic agent, a therapeutic-diagnostic agent, or some other drug of interest. The linker between the lipid moiety and the drug of interest can be cleaved in vivo (e.g., by enzymes, pH, etc.). The lipid-conjugated drug can be a prodrug, such that its release / cleavage from the lipid moiety is activated to a therapeutic or therapeutic-diagnostic form, or it can be released as a prodrug, but subsequently converted (e.g., biochemically) to its active form. Suitable lipids and linkers (e.g., cleavable and non-cleavable linkers) are known, for example as described in WO 2020 / 191477.

[0069] <Preparation of nanoparticles>

[0070] Hybrid lipid nanoparticles can be prepared using various suitable methods, such as the rapid mixing / solvent (e.g., ethanol) dilution method.Non-limiting examples of preparation methods are described in Jeffs, LB et al., Pharm Res, 2005, 22(3):362-72; and Leung, AK et al., The Journal of Physical Chemistry.C, Nanomaterials and Interfaces, 2012, 116(34):18440-18450, each of which is incorporated herein by reference in its entirety.

[0071] To incorporate a drug into the aqueous portion of the lipid nanoparticle, a loading buffer should be introduced into the lipid nanoparticle to allow the drug to proceed. There are several possible ways to introduce the loading buffer into the LNP. One possible method involves incorporating the loading buffer into the aqueous medium containing the inorganic particles during the mixing step with the lipid in the solvent (e.g., ethanol). However, aqueous dispersions of colloidal metals, such as gold, can be sensitive to ionic strength. In some embodiments, the introduction of a buffer during the mixing step can cause precipitation of the LNP. To address this possibility of LNP precipitation when the loading solution is introduced during mixing, the loading solution can be added to the LNP containing the encapsulated metal following its formation, followed by the incorporation of the loading solution into the lipid nanoparticle.

[0072] In such an embodiment, the method includes entrapping inorganic particles in lipid nanoparticles to produce lipid nanoparticles containing entrapped inorganic particles (e.g., metal nanoparticles). This includes combining a first preparation of lipids dissolved in a solvent (e.g., ethanol or other suitable solvent) and a second preparation of an aqueous solution of inorganic particles in two separate streams. The two streams are combined in a suitable mixing device to produce a merged stream, thereby forming lipid nanoparticles with entrapped inorganic particles. A loading buffer is then added to the external solution of the lipid nanoparticles thus formed. The external solution includes the solvent (e.g., ethanol or other suitable solvent) used to form the LNP containing the inorganic particles, and such a solvent can promote the incorporation of the loading buffer into the aqueous portion of the lipid nanoparticles by increasing the permeability of the LNP lipid layer, thereby entrapping the loading buffer in the lipid nanoparticles, thus producing lipid nanoparticles containing inorganic particles and the loading buffer entrapped in its interior or interior compartment. To create a transmembrane chemical gradient, the original external medium of the lipid nanoparticles is replaced with a new external medium having a different concentration of species (e.g., protons) that drive loading. The method then includes introducing an actively loadable drug into the external solution of the lipid nanoparticles containing the entrapped loading buffer, allowing the drug to be actively loaded into the lipid nanoparticles, thereby producing lipid nanoparticles that entrap both the inorganic particles and the actively loadable drug.

[0073] Exchange of the external medium can be accomplished by a variety of techniques, such as passing the lipid nanoparticles through a gel filtration column, such as a Sephadex column, equilibrated with new medium (as shown in the examples below), or by centrifugation, dialysis, or related techniques. In the case of pH gradient loading, the internal medium can be either acidic or basic relative to the external medium. In such embodiments, after establishment of the pH gradient, a pH gradient loadable drug is added to the mixture and encapsulation of the drug into the lipid nanoparticles occurs as described above.

[0074] Loading using a pH gradient can be carried out according to known methods, for example as described in US Pat. Nos. 5,616,341, 5,736,155, and 5,785,987, each of which is incorporated herein by reference.

[0075] Although pH gradient loading has been described, active loading involves the use of any suitable transmembrane chemical gradient across the LNP membrane to trigger the uptake of an actively loadable agent after the LNP is formed. + , K + , H + and / or a gradient of protonated nitrogen moieties, including one or more ions, including: In other words, active loading techniques that may be used in accordance with the present disclosure include, but are not limited to, pH gradient loading, charge attraction, and drug shuttling with an agent capable of binding to the drug.

[0076] <Nanoparticle morphology>

[0077] The lipid nanoparticle comprises a core that encapsulates both inorganic particles and a drug, which in some embodiments is a therapeutic or imaging agent. The term "core" or "inner core" refers to a trapped or at least partially enclosed spatial region of a lipid nanoparticle that includes an aqueous portion and optionally an electron-dense region (e.g., a hydrophobic core). The aqueous portion and the electron-dense region, if present, can be visualized by cryo-EM microscopy. In one embodiment, at least about 1 / 4 of the core contains an aqueous portion, or at least about 1 / 3 of the core contains an aqueous portion, or at least about 1 / 2 of the core contains an aqueous portion, as qualitatively determined by cryo-EM or other suitable techniques. In one embodiment, the shape of the lipid nanoparticle is circular or elongated in cross section.

[0078] This unique morphology may depend on the proportion of "bilayer" lipids (helper lipids) in the lipid nanoparticles. It has been observed that as the helper lipid (e.g., DSPC) increases, the helper lipid first forms a monolayer around the core region, which is hydrophobic, and then forms a bilayer surrounding the core. As the proportion of ionizable lipids is further decreased, the size of the hydrophobic regions decreases and the number of lamellae increases. Without intending to be limited by theory, it may be proposed that LNPs containing negatively charged inorganic particles at maximum inorganic particle (e.g., gold nanoparticles (GNPs)) capture levels represent a limiting situation in which there are essentially no hydrophobic regions in the core, and the inorganic particles are complexed or associated with ionizable lipids to locate at the intersections of the lamellae.

[0079] In one embodiment, the lipid nanoparticle surface is substantially uncharged as determined by measuring the zeta potential of the LNPs described herein. This may be due to an outer lipid layer (e.g., bilayer) having low levels of ionizable lipids and high helper lipid content. In alternative embodiments, the LNPs are unilamellar or multilamellar.

[0080] Without wishing to be bound by theory, inorganic particles may be associated or complexed with ionizable lipids. In some embodiments, encapsulated inorganic particles are present in the region of the particle where two lipid layers meet in the lipid nanoparticle, as detected by cryo-TEM microscopy. However, it is understood that the present invention is not limited by the location or nature of the incorporation of inorganic particles in the lipid nanoparticle. That is, the term "encapsulated" is not meant to be limited to any particular interaction between inorganic particles and lipid nanoparticles. Inorganic particles may be incorporated in the aqueous portion, either lipid layer, or both.

[0081] <Pharmaceutical preparations>

[0082] In some embodiments, the present disclosure provides a method for treating or imaging cells of a subject by administering at least one lipid nanoparticle to the cells in vivo. The method for treating or imaging cells may further include applying an external energy source, such as a light source, a laser (continuous wave (cw) or pulsed), X-rays, or gamma rays. The energy source causes at least partial release of the contents of the lipid nanoparticles to enable imaging and / or therapeutic effects. As known to those skilled in the art, irradiating lipid nanoparticles with a suitable energy source increases the rate of degradation of the lipid nanoparticles.

[0083] In some embodiments, the lipid nanoparticles of the present disclosure are part of a pharmaceutical composition and are administered to treat and / or prevent a disease state. The treatment may provide prophylactic (preventive), ameliorative, or therapeutic benefits. The pharmaceutical composition is administered in any suitable dosage. In one embodiment, the pharmaceutical composition is administered parenterally, i.e., intraarterially, intravenously, subcutaneously, or intramuscularly. In yet a further embodiment, the pharmaceutical composition is for intratumoral or intrauterine administration. In another embodiment, the pharmaceutical composition is administered intranasally, intravitreally, subretinal, intrathecal, or via other topical routes.

[0084] The pharmaceutical composition may further comprise one or more pharma- ceutically acceptable excipients. Excipients are substances that are included in pharmaceutical compositions to stabilize for long periods of time, bulk solid formulations containing small amounts of potent active ingredients (i.e., they may function as "bulking agents," "fillers," or "diluents"), or in some cases to enhance the delivery of the active ingredient in the final dosage form, for example, by promoting drug absorption, reducing viscosity, or modifying (often increasing) solubility. Excipients may also play a role in facilitating / improving manufacturing, for example, acting as anti-adherents, binders, coatings, glidants, lubricants, preservatives, adsorbents, and / or vehicles (for liquid and gel formulations). The term excipient encompasses the terms "carrier" and "diluent." Non-limiting examples of suitable excipients include any suitable buffer, stabilizer, salt, antioxidant, complexing agent, isotonicity agent, cryoprotectant, lyoprotectant, suspending agent, emulsifier, antimicrobial agent, preservative, chelating agent, binder, surfactant, wetting agent, non-aqueous vehicle such as fixed oil, or polymer for sustained or controlled release. See, for example, Berge et al., 1977 (J.Pharm Sci. 66:1-19), or Remington-The Science and Practice of Pharmacy, 21st ed. (Gennaro et al., editors, Lippincott Williams & Wilkins, Philadelphia), each of which is incorporated by reference in its entirety. As used herein, "pharmaceutical acceptable" refers to a substance that is acceptable for use in pharmaceutical applications from a toxicological standpoint and has no deleterious interactions with the active ingredient. Thus, a pharmaceutically acceptable carrier is one that is compatible with other ingredients in the formulation and biologically acceptable. Supplementary active ingredients may also be incorporated into the pharmaceutical composition. Excipients include, but are not limited to, binders, fillers, flow aids / glidants, disintegrants, lubricants, stabilizers, surfactants, and the like.

[0085] The compositions described herein can be administered to a subject. As used herein, the terms "patient" and "subject" include human or non-human subjects. As used herein, the terms "treat," "treatment," "therapeutic," and the like, include ameliorating symptoms, reducing disease progression, improving prognosis, and reducing recurrence.

[0086] In some embodiments, lipid nanoparticles have a long circulation life, which may be beneficial for reaching extrahepatic tissues. In some embodiments, lipid nanoparticles have improved scalability. In some embodiments, lipid nanoparticles improve the internalization of GNPs (or other metal nanoparticles) to avoid immune response issues. In some embodiments, lipid nanoparticles have an improved ability to efficiently encapsulate drug cargo.

[0087] Triggered release of one or more loaded drugs of interest

[0088] Lipid nanoparticles can be designed to accumulate in the tumor microenvironment, where they release the encapsulated therapeutic agent only in the target cells, e.g., cancerous cells. Furthermore, increased vascular permeability of lipid nanoparticles (≦200 nm) due to the well-known Enhanced Permeability and Retention (EPR) effect leads to increased accumulation in the tumor microenvironment, thereby minimizing the undesirable side effects of chemotherapy (Greish, 2010, Methods in Molecular Biology, vol. 624, pp. 25-37; Jhaveri and Torchilin, April 25, 2014, Frontiers in Pharmacology, Review vol. 5, no. 77, pp. 1-26).

[0089] Lipid nanoparticles with long-term stability (e.g., no or limited cargo leakage under normal physiological conditions) can be designed to provide controlled sustained release of encapsulated cargo by stimuli-activated approaches in the tumor microenvironment. Such internal stimuli-responsive delivery systems are advantageous in that they can destabilize lipid nanoparticles (i.e., by degrading lipid nanoparticle components) in response to changes in cellular pH, redox conditions, enzymes, and / or temperature at the target site (Simoes et al., 2001, Biochimica et Biophysica Acta (BBA)-Biomembranes, vol. 1515, no. 1, pp. 23-37; Li et al., 2015, Asian Journal of Pharmaceutical Sciences, vol. 10, no. 2, pp. 81-98; Yatvin et al., 1978, Science, vol. 202, no. 4374, pp. 1290-3).

[0090] In contrast to internally stimulated release systems, externally responsive systems (e.g., using magnetic, ultrasonic, thermal, microwave, radiofrequency, or light stimuli) have the potential to release encapsulated cargo in a target microenvironment, independent of differences in cellular mechanisms / phenomena between target and non-target sites (Chander et al., 2021, Small, vol. 17, no. 21, 2008198; Mathiyazhakan et al., 2014, Colloids and surfaces. B, Biointerfaces, vol. 126, pp. 569-574). Examples include mild, local hyper-thermic trigger methods (e.g., ThermoDox™ technology), as well as phototherapy and photodynamic therapy.

[0091] Laser-induced drug release from lipid nanoparticles, i.e., simply by focusing a laser beam on the target tissue (deep inside the body), can ensure a rapid transition temperature change without damaging surrounding non-cancerous cells. This form of photoinduced drug release utilizes pulsed lasers (e.g., with femto, pico, or nanosecond pulses) with a peak in the near-infrared (NIR) region, and thus can deliver a stronger and more concentrated amount of energy (e.g., highly localized temperature increase of a few nanoseconds within a few nanometers without excessive tissue heating) to exclusively release the cargo, compared to other photoinduced systems. To achieve such effects, lipid nanoparticle formulation methods are adapted by incorporating plasmonic nanoparticles inside. This allows for the amplified localized electromagnetic plasma field to be specifically focused inside liposome targets to trigger drug leakage in response to pulsed laser irradiation (Boulais et al., 2012, Nano Lett, vol. 12, no. 9, pp. 4763-9; Boulais and Meunier, "Basic mechanisms of the femtosecond laser interaction with a plasmonic nanostructure in water", Proceedings of SPIE-The International Society for Optical Engineering, vol. 7925, DOI: 10.1117 / 12.876193; Patskovsky et al., 2014, The Analyst, vol. 139, pp. 5247-5253). The strong and rapid response of plasmonic nanoparticles to incident light, on / off resonant irradiation with optimized laser fluence, whether single shot or multi-shot (e.g., by adjusting the spatial overlap and exposure time between pulses), can facilitate controlled local energy absorption and release, allowing site-specific drug release in the tumor microenvironment without harming non-target cells (Pustovalov, 2005, Chemical Physics, 308:103-108; Pustovalov et al., Laser Physics Letters, 5:775-792).

[0092] The LNPs disclosed herein can be triggered by a variety of stimuli to release one or more loaded agents encapsulated in the LNP, resulting in a more targeted release of the loaded agent, resulting in increased efficacy and / or reduced adverse effects. Common stimuli include, but are not limited to, light (electromagnetic radiation), magnetic field, temperature, ultrasound, pH, oxidation-reduction, or biochemical stimuli (e.g., enzymes, etc.).

[0093] In one embodiment, an LNP or composition disclosed herein is administered to a subject and the microenvironment of the target tissue (e.g., a tumor) triggers the release of one or more loaded agents, such as anti-cancer agents, therapeutic diagnostic agents, or tumor imaging agents.

[0094] In another aspect, the LNPs or compositions disclosed herein are administered to a subject and an external stimulus is administered (e.g., at a body target site) to release the loaded agent. Non-limiting examples of release triggered by an external stimulus are provided in Examples 6-8. For example, but not limited to, visible / infrared light can be used to trigger the release of an agent of interest from gold- or silver-containing LNPs, radio frequency radiation can be used to trigger the release of an agent of interest from gold-containing LNPs, and a magnetic field can be used to trigger the release of an agent of interest from LNPs containing iron oxide, cobalt, or nickel, or an alloy of gold with any of the aforementioned magnetic metals. In some embodiments, the inorganic particles comprise iron oxide and the external stimulus is a magnetic field. In some embodiments, the inorganic particles comprise gold and the external stimulus is electromagnetic radiation (e.g., NIR or UV). In some embodiments, the inorganic particles comprise gold or silver and the external stimulus is irradiation from a light source or irradiation by a laser. In some embodiments, the inorganic particles are metal nanoparticles (e.g., gold or silver) and the stimulus is irradiation (optionally using a laser) at a wavelength that is resonant or not resonant with the plasmon peak of the metal nanoparticles. In some embodiments, the laser is continuous wave or pulsed. In some embodiments, the laser is pulsed with microsecond, nanosecond, picosecond, or femtosecond pulse widths. In some embodiments, the irradiation is a femtosecond laser with a wavelength that is resonant or not resonant with the metal nanoparticles, and the metal nanoparticles have plasmonic properties.

[0095] A set of non-limiting embodiments are defined below. Embodiment 1. A lipid nanoparticle comprising: A helper lipid and an ionizable lipid, the ionizable lipid being present at 2 mol% to 30 mol% of the total lipid; at least one lipid layer surrounding an inner core, the inner core having at least one aqueous portion; Encapsulated inorganic particles; a drug of interest, the drug being a hydrophilic drug present in at least one aqueous portion or a lipophilic drug present in at least one lipid layer; A lipid nanoparticle comprising: Embodiment 2. The lipid nanoparticle of embodiment 1, wherein the drug of interest is hydrophilic and is present in at least one aqueous portion. Embodiment 3. The lipid nanoparticle of embodiment 2, wherein the drug of interest is precipitated in at least one aqueous portion. Embodiment 4. The lipid nanoparticle of embodiment 2 or 3, wherein at least one aqueous portion is acidic and the drug of interest is a weak base. Embodiment 5. The lipid nanoparticle of embodiment 2 or 3, wherein at least one aqueous portion is basic and the drug of interest is a weak acid. Embodiment 6. A lipid nanoparticle according to embodiment 1, wherein the drug of interest is lipophilic and is present in at least one lipid layer. Embodiment 7. The lipid nanoparticle of embodiment 1, wherein the drug of interest is a hydrophilic drug conjugated to a lipid moiety by a cleavable linker and is present in at least one lipid layer. Embodiment 8. A lipid nanoparticle according to any one of embodiments 1 to 7, wherein the inorganic particles present in the core are metal nanoparticles. Embodiment 9. A lipid nanoparticle according to any one of embodiments 1 to 8, wherein the inorganic particles comprise gold or iron. Embodiment 10. A lipid nanoparticle according to any one of embodiments 1 to 9, wherein the inorganic particles present in the core are colloids. Embodiment 11. A lipid nanoparticle according to any one of embodiments 1 to 10, wherein the inorganic particles present in the core have a diameter of 1 to 20 nm. Embodiment 12. A lipid nanoparticle according to any one of embodiments 1 to 11, having an average diameter of 50 to 200 nm. Embodiment 13. The lipid nanoparticles of claim 12 having an average diameter of 70 to 150 nm. Embodiment 14. A lipid nanoparticle according to any one of embodiments 1 to 13, wherein at least one lipid layer is a bilayer (two-layer) or multilayer. Embodiment 15. The lipid nanoparticle of embodiment 14, wherein the helper lipid is a neutral lipid and the lipid component of the nanoparticle further comprises at least one of cholesterol and a hydrophilic polymer-lipid conjugate. Embodiment 16. Lipid nanoparticles according to any one of embodiments 1 to 15, wherein the helper lipid is present in a concentration of at least 20 mol%, optionally at least 30 mol%, or optionally 40 mol%. Embodiment 17. A lipid nanoparticle according to any one of embodiments 1 to 16, wherein the ionizable lipid is present at 5 mol% to 15 mol% relative to the total lipid. Embodiment 18. A lipid nanoparticle according to any one of embodiments 1 to 17, wherein a pH gradient exists across the lipid layer. Embodiment 19. A lipid nanoparticle according to any one of embodiments 1 to 18, wherein the inorganic particles are negatively charged. Embodiment 20. A lipid nanoparticle according to any one of embodiments 1 to 19, wherein the inorganic particles comprise a negatively charged cap. Embodiment 21. A lipid nanoparticle according to any one of embodiments 1 to 20, wherein the ionizable lipid is cationic at physiological pH. Embodiment 22. A lipid nanoparticle according to any one of embodiments 1 to 21, wherein the inorganic particles are complexed with ionizable lipids and the inorganic particles are located at the intersections of the lamellae of the lipid layer. Embodiment 23. A method for producing lipid nanoparticles entrapping inorganic particles and a drug of interest, comprising: (i) combining a first preparation of lipids dissolved in a solvent and a second preparation of an aqueous solution of inorganic particles in two separate streams to produce a combined stream, thereby forming lipid nanoparticles encapsulating inorganic particles in the combined stream; (ii) introducing a loading medium into the solvent-containing exterior solution of the lipid nanoparticles thereby formed, and entrapping the loading medium in the lipid nanoparticles, thereby producing lipid nanoparticles comprising inorganic particles and entrapped loading medium in their interior compartments; (iii) introducing a drug of interest into the external solution of the lipid nanoparticles containing the entrapped loading buffer, and allowing the drug of interest to be actively loaded into the lipid nanoparticles in response to the entrapped loading medium, thereby producing lipid nanoparticles having entrapped inorganic particles and the drug of interest; A method comprising: Embodiment 24. The method of embodiment 23, wherein the pH of the aqueous solution of inorganic particles in step (i) is less than 5.5. Embodiment 25. The method of embodiment 23 or 24, wherein the lipid dissolved in the first preparation comprises an ionizable lipid, optionally a cationic lipid. Embodiment 26. The method of embodiment 25, wherein the ionizable cationic lipid is an amino lipid and the pH of the aqueous medium is less than the pKa of the ionizable cationic lipid, such that the ionizable cationic lipid is charged. Embodiment 27. The method of any one of embodiments 23 to 26, wherein the first and second preparations are pumped and mixed in a "T" mixer. Embodiment 28. The method according to any one of embodiments 23 to 27, wherein the external solution of the lipid nanoparticles is exchanged with a solution having a pH at least 1 pH unit higher than the pH of the loading buffer. Embodiment 29. The method of embodiment 28, wherein the loading medium is added to the lipid nanoparticles before the external solution is exchanged. Embodiment 30. The method of any one of embodiments 23 to 29, wherein the solvent in the first preparation is ethanol. Embodiment 31. The method according to any one of embodiments 23 to 30, wherein the lipid in the first preparation comprises a helper lipid and an ionizable lipid, and the ionizable lipid is present at 2 mol% to 30 mol% of the total lipid, optionally at 5 mol% to 15 mol% of the total lipid. Embodiment 32. The method of embodiment 31, wherein the helper lipid is present in a concentration of at least 20 mol%, optionally at least 30 mol%, optionally at least 40 mol%. Embodiment 33. A method for producing lipid nanoparticles comprising an inorganic particle core and a lipophilic drug of interest, comprising: combining in two separate streams a first preparation of lipids dissolved in a solvent and a second preparation of an aqueous solution of inorganic particles to produce a combined stream, thereby forming lipid nanoparticles in the combined stream that encapsulate inorganic particle cores; the lipids in the first preparation include a helper lipid, an ionizable lipid, and a lipophilic drug of interest; The ionizable lipid is present at 2 mol % to 30 mol % of the total lipid, and optionally at 5 mol % to 15 mol % of the total lipid; and A method wherein the helper lipid is present in a concentration of at least 20 mol %, optionally at least 30 mol %, optionally at least 40 mol %. Embodiment 34 The method of embodiment 33, wherein the agent of interest is a hydrophilic agent of interest conjugated to a lipid moiety. Embodiment 35. The method of embodiment 34, wherein the lipid moiety is conjugated to the hydrophilic drug of interest via a cleavable linker. Embodiment 36. The method according to any one of embodiments 23 to 35, wherein the lipid nanoparticles produced by the method are as defined in any one of claims 1 to 22. Embodiment 36. Use of the lipid nanoparticles described in any one of embodiments 1 to 22 for delivering a desired agent to a subject, wherein the lipid nanoparticles are administered to a subject and subsequently a stimulus is administered to an area of ​​the subject, the stimulus causing inorganic particles in the lipid nanoparticles to release the desired agent from the lipid nanoparticles, optionally the stimulus being electromagnetic radiation from a light source or laser. Embodiment 37. The use according to embodiment 36, wherein the agent of interest is a therapeutic agent for treating a disease or condition of a subject. Embodiment 38. The use of embodiment 36, wherein the agent of interest is an imaging agent and the use further comprises imaging an area of ​​the subject. Embodiment 39. The use of embodiment 36, wherein the agent of interest is a prodrug comprising a lipophilic therapeutic agent conjugated to a lipid moiety via a cleavable linker, and the use further comprises causing cleavage of the cleavable linker, and after cleavage, the therapeutic agent treats a disease or condition of the subject. Embodiment 40. A lipid nanoparticle, comprising: An ionizable lipid content of 2 mol% to 30 mol%; at least one of a hydrophilic polymer-lipid conjugate and a sterol; A helper lipid content of more than 30 mol% that forms a bilayer surrounding the aqueous portion; Inorganic particles present in the bilayer; a therapeutic agent and / or an imaging agent present in the aqueous portion; A lipid nanoparticle, wherein the therapeutic agent and / or imaging agent is releasable from the lipid nanoparticle upon irradiation. Embodiment 41. The lipid nanoparticle of embodiment 40, wherein the therapeutic or imaging agent is precipitated in the aqueous portion. Embodiment 42. A lipid nanoparticle according to embodiment 40 or 41, wherein the inorganic particles are metal nanoparticles and comprise gold or iron. Embodiment 43. The lipid nanoparticle of embodiment 42, wherein the metal nanoparticles present in the aqueous portion are colloidal. Embodiment 44. A lipid nanoparticle according to any one of embodiments 40 to 43, having an average diameter of 50 to 200 nm. Embodiment 45. The lipid nanoparticles of embodiment 44 having an average diameter of 70 to 150 nm. Embodiment 46. The lipid nanoparticle of embodiment 45, wherein the metal nanoparticles present in the bilayer have a diameter of 1 to 20 nm. Embodiment 47. A lipid nanoparticle according to any one of embodiments 40 to 46, wherein the helper lipid is a phosphatidylcholine lipid. Embodiment 48. A lipid nanoparticle according to any one of embodiments 40 to 47, wherein the helper lipid is present in a concentration of at least 35 mol%. Embodiment 49. A lipid nanoparticle according to any one of embodiments 40 to 47, wherein the helper lipid is present in a concentration of at least 40 mol%. Embodiment 50. A lipid nanoparticle according to any one of embodiments 40 to 47, wherein the helper lipid is present in a concentration of at least 45 mol%. Embodiment 51. A lipid nanoparticle according to any one of embodiments 40 to 50, wherein the cationic lipid is present at 5 mol% to 15 mol% relative to the total lipid. Embodiment 52. A lipid nanoparticle described in any one of embodiments 40 to 51, wherein a pH gradient exists across the lipid layer. Embodiment 53. The lipid nanoparticle of embodiment 52, wherein the aqueous core is acidic and the solution outside the lipid nanoparticle is basic. Embodiment 54. A lipid nanoparticle described in any one of embodiments 40 to 53, wherein the therapeutic agent is a weak base and is capable of pH gradient loading. Embodiment 55. A lipid nanoparticle according to any one of embodiments 40 to 54, wherein the metal nanoparticles are negatively charged. Embodiment 56. A lipid nanoparticle according to any one of embodiments 40 to 55, wherein the metal nanoparticle comprises a negatively charged cap. Embodiment 57. A lipid nanoparticle according to any one of embodiments 40 to 56, wherein the ionizable lipid is cationic at less than physiological pH. Embodiment 58. A method for producing lipid nanoparticles according to any one of embodiments 1 to 22 or 40 to 57, comprising an ethanol mixing method. Embodiment 59. Use of lipid nanoparticles according to any one of embodiments 1 to 22 or 40 to 57 for treating a mammalian subject in need thereof, comprising triggered release of a drug at a body target site. Embodiment 60. The use of lipid nanoparticles according to embodiment 59, wherein the triggered release is caused by irradiation from a light source or by irradiation with a laser. Embodiment 61. The use of lipid nanoparticles according to embodiment 60, wherein the irradiation has a wavelength that is resonant or not resonant with the plasmon peak of the metal nanoparticles. Embodiment 62. The use of lipid nanoparticles according to embodiment 61, wherein the induced emission is triggered by a laser and is resonant. Embodiment 63. The use of lipid nanoparticles described in embodiment 61, wherein the laser is continuous wave or pulsed. Embodiment 64. The use of lipid nanoparticles described in embodiment 61, wherein the laser is pulsed with a pulse width of microseconds, nanoseconds, picoseconds, or femtoseconds. Embodiment 65. The use of lipid nanoparticles described in embodiment 61, wherein the irradiation is a femtosecond laser having a wavelength that is resonant or not resonant with the metal nanoparticles, and the metal nanoparticles have plasmonic properties. Embodiment 66. A method of medical treatment comprising administering lipid nanoparticles described in any one of embodiments 1-22 or 40-57 to a mammalian subject in need of such treatment, and subjecting the lipid nanoparticles to irradiation to induce release of a drug at a body target site. Embodiment 67. The method of embodiment 66, wherein the emission is triggered by irradiation from a light source or by irradiation with a laser. Embodiment 68. The method of embodiment 66, wherein the irradiation has a wavelength that is resonant or not resonant with the plasmon peak of the metal nanoparticles. Embodiment 69. The method of embodiment 66, wherein the stimulated emission is caused by a laser and is resonant. Embodiment 70. The method of embodiment 66, wherein the laser is continuous wave or pulsed. Embodiment 71. The method of embodiment 66, wherein the laser is pulsed with a microsecond, nanosecond, picosecond, or femtosecond pulse width. Embodiment 72. The method of embodiment 66, wherein the irradiation is a femtosecond laser having a wavelength that is resonant or not resonant with the metal nanoparticles, and the metal nanoparticles have plasmonic properties.

[0096] The following examples are intended to illustrate the preparation and properties of specific lipid nanoparticle preparations, but are not intended to limit the scope of the invention in any way. EXAMPLES

[0097] <Material>

[0098] Lipids (1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy-(polyethylene glycol)-2000] (PEG-DSPE), and the ionizable cationic lipid 1,2-dioleoyl-3-dimethylammonium-propane (DODAP)) were purchased from Avanti Polar Lipids (Alabaster, AL). Cholesterol (Chol), sodium acetate, ammonium sulfate (AS), and doxorubicin hydrochloride were obtained from Sigma-Aldrich Canada Ltd. (Oakville, ON, Canada). Phosphate-buffered saline was obtained from GIBCO (Carlsbad, CA). Dialysis membranes (molecular weight cutoff 12,000–14,000 Da) were obtained from Spectrum Laboratories (Rancho Dominguez, CA). Amicon Ultracel centrifugation units (10 kDa MWCO) were obtained from Millipore (Billerica, MA). Tannic acid stabilized negatively charged monodisperse spherical gold nanoparticles (5 nm diameter, 5.5×1013 particles / ml) were provided in the form of an aqueous dispersion by Ted Pella, Inc. (Redding, CA, USA). Anion exchange spin columns (Vivapure D Mini H) were obtained from Sartorius Stedim Biotech, Aubagne, France. QuantiFluo™ Fluorescent Ammonia Measurement Assay Kit was obtained from BioAssay Systems (Hayward, CA). Cholesterol E Total Cholesterol Assay Kit was provided by Wako Diagnostics (Richmond, VA).

[0099] <Method>

[0100] <Synthesis of gold / lipid hybrid nanoparticles> LNP-GNPs were prepared by a modified ethanol mixing method using a T-junction (Hirota et al., Biotechniques 1999, 27(2), 286-90; incorporated herein by reference). Briefly, lipid nanoparticle-gold nanoparticle particles (LNP-GNPs) were formulated by mixing an appropriate volume of lipid stock solution dissolved in ethanol buffer with an aqueous phase containing gold nanoparticles (GNPs) using a T-junction mixer. Lipids (DODAP, DSPC, Chol, and PEG-DSPE) were solubilized in a molar ratio of 10 / 49 / 40 / 1 to a final lipid concentration of 10 mg / ml in 100% ethanol. Aqueous gold colloids (nominal particle size 5 nm, particle concentration 5.5 mg / ml, supplied by the manufacturer) were concentrated using a centrifugal concentrator, and then an appropriate volume of the concentrated dispersion was dissolved in 25 mM sodium acetate buffer (pH 4.0) to obtain the desired level of GNP number density. Acidification of the aqueous medium was necessary to fully protonate (positively charge) the cationic lipids and promote their association with the negatively charged tannic acid caps of the GNPs. Lipids dissolved in ethanol and an aqueous dispersion of GNPs were pumped by two syringe pumps (volume flow ratio 3:1 (aqueous dispersion to ethanol), pump speeds of 15 ml / min and 5 ml / min, respectively) and mixed at a "T" junction where the two syringes containing the organic and aqueous streams were connected to a combined connector (1 / 16'', through hole 0.02 inch, IDEX Health&Science Part#P-712). Lipids were combined with the GNPs at various gold / lipid (Au / L) ratios ranging from 1.1×1013 to 8.8×1013 particles / μmol lipid. After mixing, the gold / lipid dispersion was either dialyzed against formulation buffer to remove ethanol or dialyzed against 1× phosphate buffered saline, pH 7.4, to remove residual ethanol and neutralize the buffer. Once dialysis was completed, the average diameter size along with the ζ-potential of the spontaneously formed LNP-GNPs was measured (Zetasizer Nano ZS, Malvern Instruments Inc., Westboro, MA). Lipid concentration was determined by measuring total cholesterol using an enzymatic assay.Before and after removing uncaught gold using an anion exchange spin column, the absorbance at 520 nm (the maximum absorbance for 5 nm spherical GNPs) was measured in the recovered samples, and colloidal gold was quantified by comparing the respective Au / L ratios, thereby measuring the GNP capture efficiency. Absorbance measurements were performed with 1% Triton X-100 at the time of dissolution of LNP-GNP and release of the captured gold nanoparticles.

[0101] <Synthesis of Gold / Lipid Nanoparticle Systems Capable of Remote Filling of GNPs>

[0102] To realize an LNP-GNP system capable of remotely filling and stably retaining a drug cargo, a freshly prepared lipid / gold mixture (prepared as above) dispersed in an acetate buffer containing 25% ethanol was added to a concentrated solution of ammonium sulfate (AS). Briefly, 1 ml of an AS aqueous solution (typically 0.9, 1.35, and 1.8 M) was added dropwise to 2 ml of vortexed lipid / gold dispersion, and then the resulting mixture was placed in a dialysis bag and dialyzed against phosphate buffered saline to remove ethanol and uncaught AS and raise the pH to 7.4. This procedure results in LNP-GNPs that capture AS in an amount sufficient to effect uptake and stable retention of an externally added drug by the active filling mechanism 27. The rate of AS capture was determined by measuring the ammonium concentration in the samples recovered before and after dialysis (i.e., before and after removal of uncaught ammonium) using a fluorescent ammonia / ammonium measurement assay kit. Measurements were performed in the presence of 1% Triton X-100 to dissolve the LNP-GNP and release its contents. Particle size and lipid concentration were measured as described above.

[0103] <Filling of Therapeutic Agents into LNP-GNPs>

[0104] Before filling, the ammonium sulfate-containing LNP-GNP system was concentrated to about 10 mg / ml of lipid using a centrifugal concentrator. Doxorubicin hydrochloride was dissolved in physiological saline at 5 mg / ml and mixed with the LNP-GNP dispersion to obtain the desired drug / lipid (D / L) ratio. The sample was then incubated at 60 °C to optimize the filling conditions. Uncaptured doxorubicin was removed by flowing the sample through a Sephadex G-50 spin column before detecting the captured drug. Doxorubicin was assayed by fluorescence intensity (excitation wavelength 480 nm, emission wavelength 590 nm) using a Perkin-Elmer LS50 fluorometer (Perkin-Elmer, Norwalk, CT), and values for 100% release were obtained by adding isopropanol to a final concentration of 50 vol%. The drug loading efficiency was determined by quantifying both the drug levels and lipid levels in the samples obtained before and after separating the uncaptured drug from the LNP-GNP encapsulated drug by size exclusion chromatography using a Sephadex G-50 spin column and comparing the respective drug / lipid ratios.

[0105] <Cryo-transmission electron microscopy (cryo-TEM) analysis of the LNP form>

[0106] Using methods previously described by Leung et al., J Phys Chem B 2015, 119(28), 8698 - 706; Witzigmann et al., Adv Drug Deliv Rev 2020, 159, 344 - 363 (which are incorporated herein by reference), the hybrid LNP - GNP system was imaged with a FEI Tecnai G20 TEM (FEI, Hillsboro, Oregon). Prior to imaging, the sample was concentrated to approximately 20 mg / mL total lipid, and 3 - 5 μl aliquots of the concentrated dispersion were transferred to glow - discharged copper grids of a FEI Mark IV Vitrobot. The sample was then plunge - frozen in liquid ethane to generate vitreous ice. The frozen samples were stored in liquid nitrogen until imaging. The TEM was operated at 200 kV in low - dose mode, and images were obtained at a nominal under - focus of 2 - 4 μm using a bottom - mounted FEI high - resolution CCD camera (FEI, Hillsboro, Oregon). Sample preparation and image acquisition were performed at the UBC Bioimaging Facility (Vancouver, British Columbia).

[0107] <Measurement of Hydrophobic Prodrug in LNP - GNP>

[0108] INT - D034 was quantified by ultra - high - performance liquid chromatography (UPLC) using a Waters® Acquity™ UPLC system equipped with a photodiode array detector (PDA). Empower™ data acquisition software version 3.0 was used (Waters, USA). Separation was performed on a Waters® Acquity™ BEH C18 column (1.7 μm, 2.1×100 mm) at a flow rate of 0.5 ml / min with a linear gradient from 80 / 20 (%A / B) to 0 / 100 (%A / B). Mobile phase A consisted of water and mobile phase B consisted of methanol / acetonitrile (1:1, v / v). This method was run for 6 minutes at a column temperature of 55 °C, and the analyte was measured by monitoring the PDA detector at a wavelength of 239 nm.

[0109] Example 1. LNPs containing high levels of helper lipids can be generated that contain inorganic particles and one or more aqueous portions.

[0110] This example shows that LNPs containing inorganic particles and having one or more aqueous parts inside can be prepared by the solvent mixing method. Such LNP morphology is observed when the ionizable lipid content is lower and the helper lipid content is higher than that used in conventional formulations to formulate nucleic acids in LNPs. In previous studies, the lipid composition, i.e., ionizable lipid / cholesterol / DSPC / PEG-lipid, in a molar ratio of 50 / 38.5 / 10 / 1.5, has been mainly used to encapsulate nucleic acids.

[0111] To demonstrate the loading of inorganic particles (gold nanoparticles (GNPs)), a lipid composition with 10 mol% of ionizable lipids (DODAP / DSPC / cholesterol / PEG-lipid 10 / 49 / 40 / 1, mol / mol) was chosen. The lipid mixture dissolved in ethanol was rapidly mixed with an aqueous solution containing negatively charged inorganic particles (GNPs) using a T-tube mixer. The aqueous solution was buffered at pH 4 to protonate the ionizable lipids and thus to make them positively charged. Following the mixing step, the ethanol was removed by dialysis and the external medium was replaced by phosphate buffered saline (pH 7.4) except for the micrographs shown in Figures A and C. In Figures A and C, only the ethanol was removed and the samples were maintained at pH 4 by dialysis against 25 mM sodium acetate buffer.

[0112] As shown in Figure 1A, LNPs formed in the absence of GNPs at pH 4 (after ethanol removal by dialysis) are small (~60 nm) unilamellar vesicles, some of which are found in a "hemifused" state. In contrast, adjusting the pH to 7.4 results in "fish-eye" structures with an inner lipid core within the lipid bilayer morphology (Figure 1B). Such structures form due to high "helper" lipid content and can be accurately modeled under the assumption that the inner lipid core region corresponds to an oil core containing ionizable cationic lipids in neutral form, surrounded by a monolayer of helper lipids and PEG-lipids. Meanwhile, the surrounding bilayer can consist of or be essentially bilayer-forming helper lipids and PEG-lipids.

[0113] When GNPs were present in the formulation buffer, the particles formed at pH 4 were either unilamellar or bilamellar vesicles with a diameter of approximately 100 nm. GNPs were either localized within bilamellar vesicles or located at the interface between two unilamellar vesicles generating a "dumbbell" structure (Figure 1C, indicated by arrows). After increasing the pH to pH 7.4, the particles observed were of comparable size but almost entirely bilamellar or oligolamellar (Figure 1D-F). At an Au / L ratio of 6.6 × 10 13 At least a portion of the GNPs is localized inside the formed vesicles, up to 1 / μmol lipid. Furthermore, LNPs advantageously also contain large aqueous compartments, which offer the possibility of loading small molecule drugs.

[0114] Cryo-TEM images shown in Figure 1D-E show that the internalized GNPs are located at an apparent junction between the inner and outer vesicles containing bilayer and paucilamellar LNPs. At least a portion of the ionizable cationic lipids may also complex with the negatively charged GNPs in the same way that ionizable lipids complex with siRNA in siRNA-encapsulated LNPs. Excess ionizable lipids that do not associate with siRNA adopt a neutral oil form at pH 7.4 where they separate into hydrophobic domains. Thus, the outer lipid bilayer of the hybrid LNP-GNPs is expected to contain little or no ionizable lipids at pH 7.4 and thus exhibit no net positive surface charge. The zeta potentials of the hybrid LNP-GNP systems prepared at various Au / L ratios were measured in dilute (×0.25) PBS, as shown in Table 1. As shown, the negative ζ-potential observed for LNP-GNPs at pH 7.4 is similar to the ζ-potential obtained for the DSPC / Chol system measured under similar conditions at pH 7.4 in PBS, consistent with the suggestion that there is little or no cationic lipid on the LNP-GNP surface.

[0115] [Table 1]

[0116] As mentioned above, cryo-TEM studies of the formulations listed in Table 1 showed that 6.6 x 10 13 No evidence of free GNPs was observed for LNP-GNP systems prepared with Au / L ratios up to 8.8 × 10 13 For the system prepared with 1 / μmol lipid, clumps of aggregated GNPs are evident (Figures 1B to 1D). 13 6.6 x 10 / μmol lipid 13 Increasing the number of LNP-GNPs to 8.8 × 10 / μmol lipid did not affect the average size and surface charge properties of the LNP-GNPs. 13Further increase to 1 / μmol resulted in an increase in size and polydispersity, as well as a clear increase in net negative surface charge, in the latter case the data may reflect the coexistence of LNP-GNPs with aggregates of (negatively charged) gold particles.

[0117] [Example 2: LNP systems can be generated that contain high levels of bilayer lipids and high loadings of gold nanoparticles.]

[0118] We next evaluated the GNP encapsulation efficiency achieved for the LNP-GNP system at various Au / L ratios. Quantification of GNPs was performed by surface plasmon resonance absorption assay (Kreibig, U.; Vollmer, M., Theoretical Considerations. In Optical Properties of Metal Clusters, Springer Berlin Heidelberg: Berlin, Heidelberg, 1995; pp. 13-201; incorporated herein by reference). This is a well-established technique suitable for measuring GNPs in the presence of lipids solubilized by detergents such as Triton X-100. For that purpose, 0-5.5×10 GNPs were encapsulated in the presence of 1% Triton X-100. 13 A calibration plot in the range of 1000 / ml was prepared and the corresponding LNP GNP concentrations present in the LNP-GNP samples before and after removal of the extraneous (untrapped) gold were then calculated.

[0119] Au / L ratio of 1.1×10 after removal of untrapped gold (no surfactant added) 13 pieces~8.8×10 13 The UV-VIS absorption spectra of the LNP-GNP system at 1.1 × 10 mers / μmol lipid are shown in Figure 2 (inset). All spectra show a peak at a wavelength of approximately 520 nm, which is the fingerprint of colloidal gold nanoparticles. Quantification of the gold content revealed a concentration of 1.1 × 10 mers / μmol lipid. 13 ~6.6×10 13 Entrapment efficiencies approaching 100% were demonstrated for systems prepared with Au / L ratios in the range of 50 / μmol lipid, with Au / L ratios of 8.8 × 10 13When the concentration was increased to 100 μmol / μmol lipid, approximately 80% of the gold was internalized (Figure 3), which is in good agreement with the results of cryo-TEM studies (Figure 1B-D).

[0120] Example 3: Therapeutic agents can be actively loaded into the aqueous compartments of hybrid LNP-GNP systems.

[0121] In this example, we investigate the ability to load drugs into LNPs in addition to GNPs. The most robust procedure for drug encapsulation into LNP liposomal systems is to establish a pH gradient (acidic inside) and then load a weak base drug in response to the pH gradient. Over 50% of commonly used drugs detailed in the Merck Index are weak bases, and thus pH loading is a generally applicable procedure. An effective method to generate a pH gradient is to entrap ammonium sulfate (AS) into the vesicles during formation and then remove the external AS. Ammonium (NH4 + ) is NH3 + H + NH3 can then easily permeate to H + thus establishing a pH gradient.

[0122] [Table 2]

[0123] There are various possible ways to introduce AS into the LNPs during their formation. One method involves incorporating high concentrations of AS into the aqueous medium containing the GNPs during the mixing step with the lipids in ethanol. However, aqueous dispersions of colloidal gold are sensitive to ionic strength. It was found that adding AS solution to the GNP dispersions resulted in precipitation. An alternative approach adopted by the inventors herein was to add AS after LNP formation at pH 4. It was reasoned that the change in morphology of the LNPs as the pH increased from pH 4 to pH 7.4 (see Figures 1A and 1B) may facilitate encapsulation, and that the initial presence of 25% ethanol (v / v) may increase membrane permeability enough to allow AS to penetrate the preformed LNPs. Therefore, we added concentrated (up to 1.8 M) aliquots of AS at pH 4 to lipid dispersions containing GNPs, which were then dialyzed against PBS. (See Methods for details).

[0124] The loading study proceeded in two stages. The first stage was to determine to what extent AS could be encapsulated using a protocol in which AS was added after formulation. In this protocol, aliquots of concentrated AS were added to GNP-containing (Au / L ratio 2.2 × 10) at pH 4 to achieve final AS concentrations of 300 mM, 450 mM, and 600 mM in solution. 13 The hybrid LNPs were then dropwise added with 100% ethanol (100% / μmol) of AS. The dispersion was dialyzed against PBS to remove residual ethanol, raise the pH, and remove unentrapped AS. The resulting LNP GNP system was then solubilized in the presence of detergent and assayed for ammonium and lipid content.

[0125] As shown in Figure 4A, levels of entrapped ammonium of 0.24-0.28 mol / mol lipid were achieved for initial AS concentrations of 300 mM-450 mM at pH 4. In terms of entrapment efficiency, these ammonium entrapment levels reflect a steady decrease in entrapment efficiency as the initial AS concentration increases (Figure 4B). Importantly, there was no substantial effect on LNP size or polydispersity due to the presence of AS during the dialysis step, with only a slight size increase observed (Table 2).

[0126] It was then demonstrated that the entrapped AS was sufficient to proceed with the loading of an actively loadable therapeutic agent, in this case a weak base drug. The representative weak base drug chosen was the anticancer drug doxorubicin. This is because doxorubicin can be loaded into the liposomal LNP system to such high levels that the drug precipitates inside the LNP and forms nanocrystals that can be easily imaged by cryo-TEM. Hybrid LNP-GNP samples (pH 7.4) containing AS were prepared as described above, an aliquot of doxorubicin solution was added, and the formulation was incubated at 60 °C using the established doxorubicin loading protocol. The time course of doxorubicin uptake into hybrid LNP GNP AS systems prepared in the presence of 300, 450, and 600 mM AS was determined using an initial drug-to-lipid ratio of 0.1 (wt / wt) and is shown in Figure 5A-B. For all samples, near 100% doxorubicin entrapment efficiency was achieved within minutes of incubation (Figure 5A).

[0127] Each accumulated doxorubicin molecule consumes a proton upon reaching the acidic interior, thus reducing the internal buffering capacity of AS. If the drug-to-lipid ratio in the initial incubation medium is too high, the buffering capacity is depleted and the encapsulation efficiency decreases. Therefore, we investigated the effect of the initial drug-to-lipid ratio (0.05, 0.1, and 0.2, wt / wt) on the encapsulation efficiency of the hybrid LNP system prepared in 450 mM AS at pH 4 stage. As shown in Figure 5B, varying the drug-to-lipid ratio from 0.05 to 0.1 effectively resulted in 100% encapsulation within 5 min of incubation. However, increasing the initial drug-to-lipid ratio to 0.2 significantly reduced the entrapment efficiency. Drug loading resulted in a slight increase in particle size as measured by DLS (Table 2).

[0128] The doxorubicin loading properties of the hybrid LNP-GNP-AS system shown in Figure 5 are comparable to those reported for established formulations of liposomal doxorubicin, e.g., Doxil™, but with the added advantage of encapsulating GNPs. At high levels of encapsulation, doxorubicin precipitates into fibrillar nanocrystals, which can be detected in cryo-TEM micrographs. Therefore, cryo-TEM studies of drug-loaded LNP-GNPs to detect such nanocrystals were performed on LNP-GNPs (Au / L 2.2 × 10) prepared in 450 mM AS (added at pH 4 step) and loaded at a drug-to-lipid ratio of 0.1 (wt / wt). 13 The study was carried out using a (mg / μmol) formulation.

[0129] As shown in Figure 6, structures reflecting precipitated drug (indicated by arrows) can be observed in the hybrid LNPs, resulting in an elongated morphology of the LNPs (when compared to the more spherical LNP structures observed in the absence of drug (see Figure 1B)). This distortion may be responsible for the slight size increase of the drug-loaded LNP-GNPs, as shown by DLS. Overall, the particles showing the formation of the characteristic drug precipitate display the characteristic "coffee bean"-like appearance previously observed for various liposomal doxorubicin formulations. Advantageously, the data indicate the presence of both gold and therapeutic agent in the same lipid nanoparticles.

[0130] Example 4: Hydrophobic prodrugs can be incorporated into the hybrid LNP-GNP system.

[0131] In this example, we investigate the ability to load hydrophobic drugs into LNPs containing GNPs. Due to the lipid-like properties of lipophilic prodrugs, they are easily loaded into the LNP system by simply mixing them with the lipid components. As an example, INT-D034, a dexamethasone prodrug, was incorporated into the LNP-GNP system. The LNP-GNP system was prepared as previously described with modifications. INT-D034, ionizable or cationic lipids, DSPC, cholesterol, and PEG-DSPE were mixed in a molar ratio of 10 / 10 / 43 / 36 / 1 in ethanol. Aqueous gold colloids (nominal particle size 5 nm, particle concentration 5.5 mg / ml, supplied by the manufacturer) were concentrated using a centrifugal concentrator, and then an appropriate volume of the concentrated dispersion was dissolved in 25 mM sodium acetate buffer (pH 4.0) to obtain the desired level of GNP number density. The lipids dissolved in ethanol and the aqueous dispersion of GNPs were pumped by two syringe pumps (volume flow ratio 3:1 (aqueous dispersion to ethanol), pump speeds of 15 ml / min and 5 ml / min, respectively) and mixed at a "T" junction where the two syringes containing the organic and aqueous streams are connected to a combined connector. The formulation was dialyzed against PBS to remove residual ethanol. The physicochemical properties of the LNPs prepared as above were subsequently characterized. Particle size was measured by dynamic light scattering using a Malvern Zetasizer Nano ZS (Malvern, UK) after buffer exchange into phosphate buffered saline. Lipid concentration was determined by measuring total cholesterol using a Cholesterol E enzymatic assay kit from Wako Chemicals USA (Richmond, VA). The entrapment of INT-D034 was determined using UPLC.

[0132] Table 3 below shows that high encapsulation efficiency is observed for both GNPs and prodrugs in the LNPs.

[0133] [Table 3] [ka]

[0134] [Example 5: Release of a desired agent (e.g., a drug) using an external stimulus]

[0135] Efficient release of a target agent (e.g., a drug) from hybrid LNPs containing GNPs and a target agent after in vitro electromagnetic irradiation-Hybrid LNP formulations containing GNPs and doxorubicin (as an exemplary target agent) are prepared as described above. The physical properties of the LNPs, such as size / polydispersity and drug encapsulation efficiency, are determined by dynamic light scattering and UPLC, respectively. The morphology of the LNPs is analyzed by cryo-TEM.

[0136] A variety of energy sources have been used to trigger drug delivery and release. GNP systems can be "exploded" in response to high-energy pulsed laser radiation, which can trigger triggered release (see, for example, Letfullin, RR; Joenathan, C.; George, TF; Zharov, VP, Laser-induced explosion of gold nanoparticles: potential role for nanophotothermolysis of cancer. Nanomedicine (Lond) 2006, 1(4), 473-80), thereby disrupting the LNP membrane or structure and promoting drug release. The desired wavelength of the light source depends on the size of the GNPs, which typically ranges from 200 nm to 1000 nm.

[0137] Breast cancer cell lines MCF-7 and MDA-MB-231 are incubated with various concentrations of LNPs (10, 50, 100 μg / ml lipid) and exposed to a light source. Cell viability is assessed using a commercially available MTS-based CellTiter 96™ AQ ueousCell viability is measured using the One Solution Cell Proliferation Assay (Promega) or the resazurin-based PrestoBlue™ assay (ThermoFisher). A decrease in cell viability compared to non-irradiated control cells indicates induced doxorubicin release.

[0138] Efficient release of a target agent (e.g., a drug) from hybrid LNPs containing GNPs and a target agent after in vivo electromagnetic irradiation-Hybrid LNP formulations containing GNPs and doxorubicin (as an exemplary target agent) are prepared as described above. The physical properties of the LNPs, such as size / polydispersity and drug encapsulation efficiency, are determined by dynamic light scattering and UPLC, respectively. The morphology of the LNPs is analyzed by cryo-TEM.

[0139] The effect of induced doxorubicin release on the antitumor efficacy of the LNP system is evaluated in a mouse xenograft model. MCF-7 or MDA-MB-231 cells are implanted subcutaneously into the hind flank of Balb / c nude mice. Tumors reach a standard size (approximately 100 mm 3 Once tumor size is reached, eight mice per treatment group are intravenously injected with three increasing doses of the hybrid LNP formulation. Electromagnetic irradiation using a high-energy pulsed laser is administered to the tumor 12-24 h after LNP injection. The desired wavelength of the light source depends on the size of the GNPs (typically ranging from 200 nm to 1000 nm).

[0140] Antitumor efficacy was assessed by measuring tumor size with calipers and data are plotted as median tumor volume as a function of time (tumor volume (mm 3 ) = Length x Width 2 ) / 2). Inhibition or reduction in tumor size compared to non-irradiated control animals indicates successful induced doxorubicin release in vivo. Repeated injections of LNPs followed by electromagnetic irradiation are evaluated for comparative antitumor efficacy.

[0141] [Example 6. Design and synthesis of LNPs loaded with pH gradient-loadable drug (Dox) and gold nanoparticles (AuNPs)]

[0142] To prepare LNPs capable of encapsulating both hydrophobic negatively charged gold nanoparticles and hydrophilic drugs, such as Dox, we used the LNP formulation DODAP / DSPC / Chol / PEG-DSPE, 10 / 49 / 40 / 1 (mol / mol). The molar amounts of ionizable lipid (DODAP) and helper phospholipid (DSPC) were selected such that the LNPs were organized into bilayers.

[0143] Dox was encapsulated in LNPs with transmembrane pH gradient and acidic interior. To facilitate this, we developed a protocol to encapsulate ammonium sulfate (AS) in the aqueous core after incorporation of AuNPs into lipid nanoparticles, thereby creating a pH gradient between the interior and exterior of the lipid nanoparticles. The method to form lipid nanoparticles follows several steps. In the first step, lipids are dissolved in ethanol. The resulting solution is then mixed in an aqueous medium containing negatively charged AuNPs at a concentration of 3:1 aqueous medium to ethanol at pH 4. This is the encapsulation step of AuNPs. An aliquot of ammonium sulfate at a high concentration of 450 mM was then added. Finally, the mixture is dialyzed against saline to increase the pH and remove the ethanol. These resulting lipid nanoparticles then exhibit an outer layer composed almost entirely of Chol, DSPC, and PEG.

[0144] The obtained LNPs were observed by cryo-TEM imaging in Figures 6A and 6B. The LNPs show a spherical shape with a diameter close to 100 nm. As shown in a schematic diagram in Figure 6C, Dox is found in the aqueous core of the LNPs, while the AuNPs are densely packed in the outer layer. The physicochemical properties of the various prepared LNPs (with or without GNPs and Dox) are shown in the table shown in Figure 6D. All the LNPs show a close diameter in the different types of samples. However, a slight increase was observed when encapsulating components such as Dox or GNPs. The resulting negative potential is mainly related to the PEG layer on the outer surface of the LNPs. This PEG layer ensures the colloidal stability of the lipid nanoparticles in solution. When comparing the different samples, the values ​​differ due to the presence of Dox, which has a positive potential, and due to the presence of gold nanoparticles, which have a negative potential due to their surface coating.

[0145] The fluorescence of Dox is a characteristic used for observation, and a portion of the emission spectrum is shown in Figure 6E, where excitation is performed at appropriate wavelengths (505-555 nm) using optical filters. The fluorescence of Dox can be observed even when encapsulated in LNPs containing non-fluorescent AuNPs. The UV-vis absorbance spectrum of LNPs in Figure 6F shows a Dox absorption peak near 500 nm and a gold nanoparticle-related peak near 540 nm, which are particularly evident in the LNP / AuNP sample. This suggests that it is possible to obtain a resonance effect on LNPs with a laser irradiating at approximately 530 nm.

[0146] [Example 7: Release of Actively Loaded Drug (Dox) from Loaded LNP / AuNP / Dox after Nanosecond Laser Irradiation]

[0147] This experiment demonstrates laser irradiation to release Dox from LNPs in the MDA231 breast cancer cell line. Several irradiation protocols were performed on the cells with incubation of various samples of LNPs and controls. These LNPs enter the cells by endocytosis when they encounter the cells. The aggregation of LNPs in the endosomes can be seen in Figure 7A by the presence of fluorescent red spots in the cells for the sample without irradiation with LNP / AuNP / Dox. When the LNPs contain AuNPs and Dox, Dox penetrates the nucleus when released from the LNPs, making the nucleus intensely fluorescent as seen in Figure 7A for the image with the irradiated sample.

[0148] Figures 7A-C show representative fluorescence images of the use of a nanosecond laser to induce the release of Dox from LNP / AuNP / Dox, compared with control experiments with LNP / Dox with and without irradiation, as well as with LNP / AuNP / Dox without irradiation. In each of these protocols, a washing step is performed to remove the LNPs present in the solution, thereby reducing the fluorescence background. Experiments were performed with different incubation times of 4 h and 15 min before the irradiation and washing steps, as well as irradiation before and after the washing step. For the control without irradiation, LNP / Dox or LNP / AuNP / Dox diluted in culture medium to a concentration of 50 μg / mL (LNP) or Dox 5 μg / mL were incubated for 4 h or 15 min according to the protocol. The effect of the time of irradiation (in the presence or absence of LNPs in the surrounding medium) was studied by applying the protocol shown in Figure 7B. After a minimum incubation time of 15 min, i.e. a time chosen to allow the LNPs to diffuse and begin to penetrate the cells, the samples were irradiated. The time at which the irradiation was performed did not affect the fluorescence intensity of the cells and therefore the amount of Dox released and reaching the nucleus. In fact, it was confirmed that all results were comparable as long as all irradiations were performed during the same 1-h period. Only the incubation time, i.e. the time between the addition of the LNPs and the washing of the cells, seems to be a significant factor influencing the amount of Dox released. Fluorescence images were taken after a waiting time of 20 h to allow the laser-induced release of Dox to diffuse from the LNPs to the nucleus of the cells.

[0149] Figure 7A shows that the laser-induced release of Dox occurs only in the presence of both AuNPs and irradiation. The images of the LNP / Dox control sample with and without irradiation are similar, the laser does not release the drug, the drug remains encapsulated in the LNPs. The drug delivery result is only obtained when there is a combination of irradiation and the presence of gold nanoparticles in the lipid nanoparticles. When both are present, it can be observed that the cell nucleus is highly fluorescent, i.e., Dox has been released from the lipid nanoparticles and reached the cell nucleus. Thus, during irradiation, the gold nanoparticles absorb the laser energy, leading to the release of the cargo contained in the LNPs.

[0150] The second protocol in Figure 7B shows that the time at which irradiation is performed (before or after washing) does not affect the amount of Dox delivered to the cell nucleus: irradiation only affects LNPs that are already inside the cell (entered the cell by endocytosis) and not LNPs that are still in solution.

[0151] Moreover, Figure 7C shows that even a short incubation time of 15 min with LNPs and cells is sufficient to allow LNPs to enter cells by endocytosis. The strong fluorescence of the nucleus, seen only with the combination of both irradiation and the presence of gold nanoparticles in the outer layer of the lipid nanoparticles, indicates that Dox has been released. Note that for all these samples, all cells are still alive, since the fluorescence of calcein AM, a marker of cell viability, can be seen. However, cells whose Dox can reach the nucleus after irradiation are in the process of cell death.

[0152] Other experiments were performed to quantify the drug delivered to the nucleus, thereby evaluating the efficiency of the laser irradiation. To do this, it is considered that the measured fluorescence intensity of the nucleus is proportional to the release of Dox. Measurements were performed on the laser-induced Dox released from LNP / AuNP / Dox, as well as on Dox-loaded LNPs and Dox-loaded LNP / AuNPs in medium at similar concentrations (Dox concentration 5 μg / mL). Figure 8 shows the fluorescence intensity normalized to the condition without irradiation for LNPs containing Dox, for various irradiation and scanning parameters. It should be noted that even under irradiation, these normalized intensities remain relatively constant in the absence of GNPs in the LNPs, confirming that GNPs play a necessary role in the release of Dox. Two laser scanning conditions were used, as shown by speeds 1 and 2, where for each LNP / AuNP / Dox there was no overlap (speed 1) and some overlap (speed 2) between successive pulses. At speed 1, a threshold of 14.2 μJ of irradiation energy was found to cause a 6-fold increase in the release of Dox from LNP / AuNP / Dox when compared to the release of LNP / Dox irradiated at this fluence. For optimization of the laser scanning parameters, speed 2 (50 μm / s and a step between scan lines of 5 μm) was used to allow a small number of laser pulses to spatially overlap each other, thereby maximizing the release of Dox. Increasing the speed to 100 μm / s and a step of 10 μm (speed 2), the pulses do not spatially overlap, causing a decrease of about 30% in the release rate. In the best conditions, irradiation of LNP / AuNP / Dox caused an 11-fold increase in emission compared to the emission of the non-irradiated LNP / Dox sample.

[0153] [Example 8: Release of actively loaded drug (Dox) from LNP / AuNP / Dox after femtosecond laser irradiation]

[0154] We also investigated the possibility of using a femtosecond laser to trigger the release of Dox from LNP / AuNP / Dox (Spitfire, 800 nm, 55 fs, repetition rate 1 kHz, spot diameter 35 μm). Compared to the ns laser at 532 nm, the fs laser irradiates at a wavelength away from the Dox absorption peak, thus minimizing the undesired photochemical effects of Dox. Furthermore, irradiation in the less absorbing biological window (Barbora et al., 2021, PLoS One, vol. 16, no. 1, e0245350-e0245350; Dabrowski et al., 2016, Coordination Chemistry Reviews vol. 325, pp. 67-101; Algorri et al., 2021, Cancers, vol. 13, no. 14, pp. 3484) provides a much greater penetration depth for in vivo applications. Irradiation at 800 nm is not resonant with the plasmon peak of 5 nm AuNPs, but as shown by Boulais et al. (Nano Lett, vol. 12, no. 9, pp. 4763-9, 2012), plasma-mediated nanobubble formation can occur, thus leading to poration of the LNP bilipid layer and release of Dox.

[0155] The irradiation protocol shown in Figure 9A (bottom) and Figure 9C is similar to that used with a nanosecond laser, and the results show that, indeed, this laser can also induce the release of Dox from LNP / AuNP / Dox. 2 At a fluence of 1000 nm and with the laser pulses spatially touching each other (i.e., with a speed of 3 cm / s and a step between each scan line of 30 μm cm), strong red fluorescence is observed in FIG. 9A. FIG. 9B shows the fluorescence intensity normalized to the condition without irradiation for LNPs containing Dox for various irradiation and scanning parameters. As can be seen in FIG. 9B, the threshold for triggering the release of Dox is 61 mJ / cm. 2 and 73 mJ / cm when the pulses are in spatial contact with each other. 2 A maximum value is obtained at 73 mJ / cm. Under these conditions, each nanoparticle receives at most one pulse. In the best conditions, 2At the lowest velocity, a 3-fold increase is observed between irradiated LNP / Dox and LNP / AuNP / Dox at a fluence of 1. With this laser, a factor of 7.5 between non-irradiated and irradiated LNP / AuNP / Dox was obtained in the best conditions.

[0156] The foregoing description is intended to illustrate embodiments of the present invention and is not intended to limit the scope of the invention in any way.

[0157] All documents cited in this disclosure are incorporated herein in their entirety. All priority documents are incorporated herein in their entirety. In the event of a conflict between a term defined herein and a term defined in a cited document, the definition in this disclosure controls.

Claims

1. A lipid nanoparticle, comprising: helper lipids and ionizable lipids, the ionizable lipids being present at 2 mol% to 30 mol% of the total lipids; at least one lipid layer surrounding an interior having at least one aqueous portion; Encapsulated inorganic particles; a drug of interest, the drug being a hydrophilic drug present in said at least one aqueous portion or a lipophilic drug present in said at least one lipid layer; A lipid nanoparticle comprising:

2. The lipid nanoparticle of claim 1, wherein the drug of interest is hydrophilic and is present in the at least one aqueous portion.

3. The lipid nanoparticle of claim 2 , wherein the drug of interest is precipitated in the at least one aqueous portion.

4. said at least one aqueous portion is acidic and said drug of interest is a weak base; or the at least one aqueous portion is basic and the drug of interest is a weak acid; Optionally, the agent of interest is capable of pH gradient loading. The lipid nanoparticle according to claim 2 or 3.

5. The lipid nanoparticle of claim 1, wherein the drug of interest is lipophilic and is present in the at least one lipid layer.

6. The lipid nanoparticle of claim 1, wherein the drug of interest is a hydrophilic drug conjugated to a lipid moiety by a cleavable linker and is present in at least one lipid layer.

7. The lipid nanoparticle of any one of claims 1 to 6, wherein the agent of interest is a therapeutic agent and / or an imaging agent.

8. The lipid nanoparticle according to any one of claims 1 to 7, having an average diameter of 50 to 200 nm, optionally 70 to 150 nm.

9. The lipid nanoparticle of any one of claims 1 to 8, wherein the at least one lipid layer is bilayer or multilayer.

10. The lipid nanoparticle of claim 9, further comprising at least one of a sterol and a hydrophilic polymer-lipid conjugate, optionally wherein the sterol is cholesterol.

11. The lipid nanoparticle according to any one of claims 1 to 10, wherein the helper lipid is a neutral lipid, optionally a phosphatidylcholine lipid.

12. The lipid nanoparticle of any one of claims 1 to 11, wherein the helper lipid is present in a concentration of at least 20 mol%, optionally at least 30 mol%, optionally at least 35 mol%, optionally at least 40 mol%, or optionally at least 45 mol%.

13. The lipid nanoparticle of any one of claims 1 to 12, wherein the ionizable lipid is present at 5 mol% to 15 mol% relative to the total lipid.

14. The lipid nanoparticle of any one of claims 1 to 13, wherein a pH gradient exists across the at least one lipid layer surrounding the interior, optionally the at least one lipid layer comprises a bilayer surrounding the interior, and optionally the aqueous portion is acidic and the solution outside the lipid nanoparticle is relatively basic.

15. The lipid nanoparticle of any one of claims 1 to 14, wherein the encapsulated inorganic particle is a colloid.

16. The lipid nanoparticle of any one of claims 1 to 15, wherein the encapsulated inorganic particles have a diameter of 1 to 20 nm.

17. The lipid nanoparticle of any one of claims 1 to 16, wherein the inorganic particle is negatively charged and optionally the inorganic particle comprises a negatively charged cap.

18. The lipid nanoparticle of any one of claims 1 to 17, wherein the ionizable lipid is cationic at or below physiological pH.

19. The lipid nanoparticle of any one of claims 1 to 18, wherein the inorganic particles are complexed with the ionizable lipids, and the inorganic particles are located at the intersections of the lipid layer lamellae.

20. the at least one lipid layer comprises a bilayer surrounding the interior, the inorganic particles being in the bilayer surrounding the interior, and / or The lipid nanoparticle further comprises an inner lipid core, and the inorganic particles are in the inner lipid core. A lipid nanoparticle according to any one of claims 1 to 18.

21. The lipid nanoparticle of any one of claims 1 to 20, wherein the encapsulated inorganic particle is a metal nanoparticle.

22. The lipid nanoparticle of any one of claims 1 to 21, wherein the encapsulated inorganic particles comprise gold, iron oxide, or hybrid gold-iron oxide.

23. The lipid nanoparticle of any one of claims 1 to 22, wherein the drug of interest is releasable from the lipid nanoparticle using an external trigger, optionally irradiation.

24. A method for producing lipid nanoparticles that entrap inorganic particles and a target drug, comprising: (i) combining a first preparation of lipids dissolved in a solvent and a second preparation of an aqueous solution of inorganic particles in two separate streams to produce a combined stream, thereby forming lipid nanoparticles in the combined stream that encapsulate said inorganic particles; (ii) introducing a loading medium into an external solution of the lipid nanoparticles formed thereby, comprising the solvent, and entrapping the loading medium in the lipid nanoparticles, thereby producing lipid nanoparticles comprising the inorganic particles and the entrapped loading medium in their internal compartments; (iii) introducing the agent of interest into the lipid nanoparticle's external solution containing the entrapped loading buffer, and allowing the agent of interest to actively load into the lipid nanoparticle in response to the entrapped loading medium, thereby producing the lipid nanoparticle with entrapped inorganic particles and the agent of interest; A method comprising:

25. 25. The method of claim 24, wherein the aqueous solution of the inorganic particles in step (i) has a pH of less than 5.

5.

26. 26. The method of claim 24 or 25, wherein the lipids dissolved in the first preparation comprise an ionizable lipid, optionally a cationic lipid.

27. 27. The method of claim 26, wherein the cationic lipid is an amino lipid and the pH of the aqueous medium is below the pKa of the cationic lipid, such that the cationic lipid is charged.

28. 28. The method according to any one of claims 24 to 27, wherein the first and second preparations are pumped and mixed in a "T" mixer.

29. 29. The method of any one of claims 24 to 28, wherein the external solution of the lipid nanoparticles is exchanged with a solution having a pH at least 1 pH unit higher than the pH of the loading buffer.

30. 30. The method of claim 29, wherein the loading medium is added to the lipid nanoparticles before the external solution is exchanged.

31. The method according to any one of claims 24 to 30, wherein the solvent in the first preparation is ethanol.

32. 32. The method of any one of claims 24 to 31, wherein the lipids in the first preparation comprise helper lipids and ionisable lipids, the ionisable lipids being present at 2 mol% to 30 mol% relative to total lipid, optionally 5 mol% to 15 mol% relative to total lipid, and optionally the helper lipids being present at a concentration of at least 20 mol%, optionally at least 30 mol%, optionally at least 35 mol%, optionally at least 40 mol%, or optionally at least 45 mol%.

33. 1. A method for producing lipid nanoparticles comprising an inorganic particle core and a lipophilic drug of interest, comprising the steps of: combining in two separate streams a first preparation of lipids dissolved in a solvent and a second preparation of an aqueous solution of inorganic particles to produce a combined stream, thereby forming lipid nanoparticles in the combined stream encapsulating said inorganic particle cores; the lipids in the first preparation include a helper lipid, an ionizable lipid, and the lipophilic drug of interest; The ionizable lipid is present at 2 mol% to 30 mol% of the total lipid, optionally at 5 mol% to 15 mol% of the total lipid; and A method wherein said helper lipid is present in a concentration of at least 20 mol%, optionally at least 30 mol%, optionally at least 35 mol%, optionally at least 40 mol%, or optionally at least 45 mol%.

34. 34. The method of claim 33, wherein the agent of interest is a hydrophilic agent of interest conjugated to a lipid moiety, optionally wherein the lipid moiety is conjugated to the hydrophilic agent of interest via a cleavable linker.

35. 35. The method according to any one of claims 24 to 34, wherein the lipid nanoparticles produced by said method are as defined in any one of claims 1 to 23.

36. 24. Use of lipid nanoparticles according to any one of claims 1 to 23 for delivering a drug of interest to a subject, said lipid nanoparticles being administered to said subject followed by administration of a stimulus to an area of ​​said subject, said stimulus causing said inorganic particles in said lipid nanoparticles to release said drug of interest from said lipid nanoparticles, optionally said stimulus being electromagnetic radiation, optionally radiation from a light source or radiation by a laser.

37. 37. The use of claim 36, wherein the agent of interest is a therapeutic agent for treating a disease or condition in the subject.

38. 37. The use of claim 36, wherein said agent of interest is an imaging agent, and said use further comprises imaging said area of ​​said subject.

39. 37. The use of claim 36, wherein the agent of interest is a prodrug comprising a lipophilic therapeutic agent conjugated to a lipid moiety via a cleavable linker, and said use further comprises causing cleavage of said cleavable linker, after said cleavage, said therapeutic agent treats a disease or condition of the subject.

40. A method for producing lipid nanoparticles according to any one of claims 1 to 23, comprising an ethanol mixing method.

41. 24. Use of lipid nanoparticles according to any one of claims 1 to 23 for treating a mammalian subject in need thereof, comprising triggered release of said agent at a body target site.

42. 42. The use according to claim 41, wherein the stimulated release is caused by irradiation from a light source or by irradiation with a laser.

43. 43. The use of claim 42, wherein the irradiation has a wavelength that is resonant or not resonant with a plasmon peak of a metal nanoparticle, and optionally the stimulated emission is caused by and resonant with the laser.

44. 44. The use of claim 43, wherein the laser is continuous wave or pulsed, optionally the laser is pulsed with a microsecond, nanosecond, picosecond, or femtosecond pulse width.

45. 45. The use according to claim 44, wherein the irradiation is a femtosecond laser having a wavelength that is resonant or not resonant with the metal nanoparticles, the metal nanoparticles having plasmonic properties.

46. A method of medical treatment comprising administering lipid nanoparticles according to any one of claims 1 to 23 to a mammalian subject in need of such treatment and subjecting said lipid nanoparticles to irradiation to induce release of said agent at a body target site, optionally said release being triggered by irradiation from a light source or by irradiation with a laser.

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