Hybrid lipid nanoparticles comprising inorganic particles and agent of interest

Abstract

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

Description

[0001] priority

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 310,065 filed on February 14, 2022 and U.S. Provisional Patent Application No. 63 / 340,678 filed on May 11, 2022, each of which is incorporated by reference in its entirety. Technical Field

[0003] The present disclosure relates to lipid nanoparticles (LNPs) and methods of making the same, which lipid nanoparticles include, inter alia, an inorganic particle and an agent of interest. Background Art

[0004] The limitation of the current therapy using small molecule therapeutic agents is that less than 0.01% of the systemically administered drugs are delivered to the target site, such as tumor tissue. Many works have been carried out to enhance the delivery to the target tissue while avoiding sensitive organs. Examples include implanted (large-scale) timed release devices for slow release of drugs or nanocarriers containing drug cargoes that preferentially accumulate in the disease site (such as tumors) for systemic (intravenous infusion) administration. The implant device also needs to be accurately placed in the tumor area, but is not limited to this. On the other hand, the nanocarriers for systemic administration require stable drug encapsulation in order to be delivered to the disease site. However, since the encapsulated drug has limited bioavailability, it can be confirmed that stable encapsulation is difficult for the release of drugs at the target site. Although the release triggered at the target site in response to external stimuli can solve this problem, the targeted release after reaching the disease site has been proven to be difficult to achieve in practice.

[0005] Lipid nanoparticles (LNPs) are recognized nanocarriers for delivering a variety of cargoes to target sites in vivo (see, for example: 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 system is a mature small molecule drug (such as anticancer drug) delivery technology, of which 9 intravenous injection injectable LNP drugs have been approved by regulatory agencies worldwide. A variety of 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 possibility of causing triggered release of contents because they can "explode" in response to high-energy pulsed laser irradiation (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 often lack one or more features necessary for clinical application.

[0006] There is a need in the art for nanoparticle formulations containing inorganic particles that provide improvements over known formulations and / or provide useful alternatives. Summary of the invention

[0007] The embodiments disclosed herein represent an improvement over previous work that encapsulates both inorganic particles and therapeutic and / or imaging agents into lipid nanoparticles (LNPs) and / or provides a useful alternative thereto.

[0008] In some embodiments, the disclosure is at least partially based on the following discovery: LNP containing low-level ionizable lipids demonstrates a unique morphology, which causes both interested reagents (e.g., therapeutic agents or diagnostic / imaging agents) and inorganic particles to be effectively encapsulated in the same lipid nanoparticle. Specifically, these hybrid LNPs comprise a kernel with an aqueous portion of a reagent that can load a high level, but simultaneously these LNPs can accommodate a high level of inorganic particles therein. Dependent on the trigger release of the LNP content, lipid nanoparticles can be applied in a wide range of clinical applications. In addition, these LNPs comprise lipid components and lamellar structures, which can make the circulation life of entering extrahepatic tissues longer.

[0009] Various embodiments relate to lipid nanoparticles comprising: a helper lipid and an ionizable lipid, wherein the ionizable lipid is present at 2 mol % to 30 mol % relative to the total lipid; at least one lipid layer surrounding an interior having at least one aqueous portion; an encapsulated inorganic particle; and an agent of interest, wherein the agent of interest is a hydrophilic agent present in the at least one aqueous portion or a lipophilic agent present in the at least one lipid layer.

[0010] Also provided is a method for producing these LNPs, which can facilitate high loading efficiency of therapeutic and / or imaging agents and is scalable.

[0011] Various embodiments relate to a method for producing lipid nanoparticles encapsulating inorganic particles and an agent of interest, the method comprising: (i) combining two separate streams of 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 encapsulating the inorganic particles in the combined stream; (ii) introducing a loading medium into the external solution of the lipid nanoparticles thus formed, the external solution comprising the solvent, and causing the loading medium to be encapsulated in the lipid nanoparticles, thereby producing lipid nanoparticles containing the encapsulated loading medium and inorganic particles in their internal compartments; and (iii) introducing the agent of interest into the external solution of the lipid nanoparticles containing an encapsulated loading buffer, and causing the agent of interest to be actively loaded into the lipid nanoparticles in response to the encapsulated loading medium, thereby producing lipid nanoparticles encapsulating the inorganic particles and the agent of interest.

[0012] Various embodiments relate to a method for producing lipid nanoparticles comprising an inorganic particle core and a lipophilic agent of interest, the method comprising: combining 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 encapsulating the inorganic particle core in the combined stream; wherein the lipids in the first preparation comprise a helper lipid, an ionizable lipid, and a lipophilic agent of interest; wherein the ionizable lipid is present at 2 mol% to 30 mol% relative to the total lipids, optionally 5 mol% to 15 mol% relative to the total lipids; and wherein the helper lipid is present at a concentration of at least 20 mol%, optionally at least 30 mol%, optionally at least 40 mol%.

[0013] Various embodiments relate to the use of the lipid nanoparticles disclosed herein for delivering an agent of interest to a subject, wherein the lipid nanoparticles are used for administration to the subject, followed by administration of a stimulus to an area of ​​the subject, which stimulus causes the inorganic particles in the lipid nanoparticles to cause the agent of interest to be released from the lipid nanoparticles.

[0014] In some embodiments, the present disclosure relates to lipid nanoparticles composed of metal nanoparticles. In certain embodiments, the lipid nanoparticles include two separate internal layers / chambers to respectively encapsulate the reagent in the aqueous core and encapsulate the metal nanoparticles in the lipid layer (e.g., bilayer). Using pulsed femtosecond and nanosecond lasers to provide a local energy distribution of a few nanoseconds within a few nanometers, the formulated liposome / plasmon nanocarrier is used to deliver the reagent (e.g., a drug that can load a pH gradient, such as Dox) to the cell by site-specific light triggering in vitro or in vivo.

[0015] 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 greater than 30 mol% to form a bilayer surrounding an aqueous portion; inorganic particles present in the bilayer; and a therapeutic agent and / or imaging agent present in the aqueous portion, wherein the therapeutic agent and / or imaging agent can be released from the lipid nanoparticle by irradiation.

[0016] Various embodiments relate to treating a subject (e.g., a mammalian subject) using lipid nanoparticles disclosed herein, comprising triggering release of an agent at a body target site. Various embodiments relate to medical treatment methods, comprising administering a lipid nanoparticle as disclosed herein to a subject (e.g., a mammalian subject) in need of such treatment; and subjecting the lipid nanoparticle to irradiation to trigger release of the agent at a body target site.

[0017] Other objects, features and advantages of the present disclosure will be apparent to those skilled in the art from the following detailed description and accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 AF show encapsulation of gold nanoparticles into hybrid LNP systems using lipid compositions DODAP / DSPC / Chol / PEG-DSPE, 10 / 49 / 40 / 1 mol / mol), as visualized by cryo-transmission. (A) LNPs formed in the absence of GNPs at pH 4. (B) LNPs formed in the absence of GNPs at pH 4 and then dialyzed with PBS. (C) Au / L 2.2×10 13 The arrow indicates the “dumbbell” of GNP bridging two vesicles. (D) At pH 7.4, Au / L 2.2×10 13 LNP-GNPs formed at 6.6×10 particles / μmol lipid, (E) at pH 7.4, Au / L 6.6×10 13 LNP-GNPs formed at 100 particles / μmol lipid, (F) at pH 7.4, Au / L 8.8×10 13 The LNP-GNPs formed at 100 particles / μmol lipid. The scale bar represents 100 nm.

[0019] Figure 2 The normalized absorbance at 520 nm (au) of a LNP-GNP formulation with lipid composition DODAP / DSPC / Chol / PEG-DSPE, 10 / 49 / 40 / 1 mol / mol, measured after removal of unentrapped gold is shown relative to the initial AuNP / lipid ratio (lipid concentration normalized to 1.25 mg / ml). The inset shows the normalized absorbance spectrum obtained from the LNP-GNP system after removal of unentrapped gold (Au / L ratio, from top to bottom: 8.8×10 13 , 6.6×10 13 , 4.4×10 13 , 3.3×10 13 , 2.2×10 13 and 1.1×10 13 particles / μmol lipid).

[0020] Figure 3Graph showing the % AuNP entrapment as a function of the initial AuNP / lipid ratio for LNP-GNP formulations with lipid composition DODAP / DSPC / Chol / PEG-DSPE, 10 / 49 / 40 / 1 mol / mol. The Au / L ratios used were: (1) 1.1×10 13 、(2)2.2×10 13 、(3)3.3×10 13 、(4)4.4×10 13 、(5)6.6×10 13 and (6)8.8×10 13 particles / μmol lipid.

[0021] Figure 4 AB are bar graphs showing ammonium sulfate (AS) entrapment within LNP-GNP formulations (DODAP / DSPC / Chol / PEG-DSPE, 10 / 49 / 40 / 1 mol / mol) prepared to contain GNPs (Au / L ratio 2.2×10 13 particles / μmol) and loaded with AS by adding AS after LNP formation at pH 4, before dialyzing against PBS to remove ethanol and raise the pH to pH 7.4. The AS concentrations in the pH 4 formulation mixtures were: (1) 300 mM; (2) 450 mM; (3) 600 mM. (A) shows the ratio of entrapped ammonium to lipid (mol / mol), and (B) shows the corresponding ammonium entrapment efficiency after dialysis against PBS.

[0022] Figure 5 AB shows the embedding (%) of doxorubicin of LNP-GNP formulations encapsulated with ammonium sulfate relative to time. (A) shows the doxorubicin loading (initial drug to lipid ratio of 0.1 wt / wt) in the LNP-GNP system prepared by dialysis of PBS in the presence of 300 mM (■), 450 mM (●) and 600 mM (▲) AS at pH 4 formulation stage. (B) shows the embedding of doxorubicin prepared by the LNP-GNP system at various drug-lipid ratios (0.05 wt / wt (■), 0.1 wt / wt (●) and 0.2 wt / wt (▲)) at pH 4 stage in the presence of 450 mM AS.

[0023] Figure 6 AF shows the design and characteristics of exemplary LNP / AuNP / Dox systems. (A) is LNP (DODAP / DSPC / Chol / PEG-DSPE, 10 / 49 / 40 / 1 mol / mol) containing AuNPs (Au / L 2.2×1013 (A) is a cryo-transmission image of a LNP / AuNP system; the shape of the LNP is similar to that of the LNP with Dox in the aqueous center. (C) is a schematic diagram of different LNP samples. Dox is located in the aqueous center, while gold nanoparticles are located in the outer layer of the LNP. (D) is a table showing the physicochemical properties of the LNP, which shows the hydrodynamic diameter, size distribution (PDI) and zeta potential (measured at the same concentration of 0.1 mg / mL Dox) of the control LNP and the LNP with and without AuNP and Dox. (E) shows the fluorescence spectra of LNP / AuNP / Dox (bottom spectrum) and LNP / Dox (top spectrum) between 570nm and 610nm when using 505-555nm excitation. Fluorescence is caused by Dox, which is natural fluorescence. (F) shows the absorption spectra of LNP / AuNP / Dox (top), LNP / Dox (2nd from the top), LNP / AuNP (2nd from the bottom), LNP (bottom) and Dox (middle). Spectra are measured under the same concentration of Dox or corresponding concentration of LNP.

[0024] Fig. 7A -C shows the release of Dox after nanosecond laser irradiation with three different protocols. Fig. 7A Fluorescence images of control LNPs (no AuNPs) and MDA-MB-231 cells treated with AuNPs are shown with and without irradiation. LNPs at a concentration of 50 μg / mL. Cell membranes were stained with calcein AM in green and Dox-related red fluorescence. Irradiation was performed with an objective lens of 4× magnification and numerical aperture NA=0.13 at an energy of 14.2 μJ, a scanning speed of 50 μm / s, and scanning lines spaced apart by 5 μm steps. The pulse width was 7.7 nanoseconds. The scheme for performing these irradiations is schematically shown below the image. Irradiation was performed before the washing phase and 4 hours after the nanoparticles were added to the cells. The imaging phase was performed 20 hours later. The control samples were subjected to exactly the same scheme (i.e., no irradiation, but the same incubation time, the same waiting time before the photographing and washing steps). Figure 7B Fluorescence images of MDA-MB-231 cells incubated with LNP / AuNP / Dox for 1 hour at a concentration of 50 μg / mL of LNP are shown. Irradiation was performed under the following conditions: Figure 1 After the washing step in Figure 2 Before the washing step in Figure 3 No irradiation. Irradiation was performed with an energy of 19.7 μJ, 50 μm / s and a step size of 5 μm. The exact protocol followed is detailed below the 3 images. Irradiation was performed twice in 2 different areas, once before and once after the washing step. Figure 7C Fluorescence images of MDA-MB-231 cells treated with control LNPs (no AuNPs) and AuNPs are shown with and without irradiation. LNPs at a concentration of 50 μg / mL. Irradiation was performed with an energy of 19.7 μJ, 50 μm / s, and a 5 μm step length. Calcein AM staining of the cell membrane was green and red fluorescence associated with Dox. The scheme illustrated below the image corresponds to irradiation occurring after cell washing. LNPs were in solution with cells for only 15 minutes.

[0025] Figure 8 Shown are the normalized fluorescence intensities of LNPs containing Dox with or without gold nanoparticles (AuNPs) after irradiation with nanosecond lasers of different energies (from 0 to 19.7 μJ) at 2 different scanning parameters (i.e., speed 1: 100 μm / s and 10 μm step size, speed 2: 50 μm / s and 5 μm step size). For samples not irradiated with LNP / Dox, the normalized value was fixed to 1. Cells were incubated for 4 hours with LNP / Dox and LNP / AuNP / Dox at a concentration of 50 μg / mL.

[0026] Fig.9A -C shows Dox release after irradiation with femtosecond laser (wavelength 800 nm, pulse width 55 fs, 1 kHz repetition rate, 35 μm spot diameter). Fig.9A Fluorescence images of MDA-MB-231 cells treated with control LNPs (no AuNPs) and AuNPs are shown with and without irradiation. Irradiation protocols are schematically shown below the images. Cells were cultivated using LNPs / Dox or LNPs / AuNPs / Dox at a concentration of 50 μg / mL. Fig. 9B The results show that the energy of the samples was varied from 0 to 73 mJ / cm 2 Figure 2. Normalized fluorescence intensity of LNPs containing Dox after irradiation with a femtosecond laser at 2 different scanning parameters (i.e., speed 1: 3 cm / s and 30 μm step size, speed 2: 6 cm / s and 60 μm step size). For samples not irradiated with LNP / Dox, the normalized value was fixed to 1. Fig. 9C A schematic diagram of the irradiation space for each speed is shown. The dots represent laser pulses and the arrows represent laser paths. DETAILED DESCRIPTION

[0027] Lipid Nanoparticles

[0028] Lipid nanoparticles (LNP) described herein comprise helper lipids and cationic lipids, and the helper lipids and cationic lipids are in the level through selecting to produce LNP, and the LNP has the form that is particularly suitable for effectively encapsulating inorganic particles (for example, metal nanoparticles) and reagents (for example, therapeutic agents or diagnostic agents), such as those forms that can be loaded by active loading methods. The helper lipids can be included to be greater than 30mol%. In another embodiment, the cationic lipids exist with 2mol% to 30mol% relative to total lipids. These LNP are particularly suitable for triggering the release of LNP contents in treatment or diagnostic applications.

[0029] In a non-limiting embodiment, a lipid layer (such as a double layer or other lamellar structure) surrounds the inside of the LNP, which includes an aqueous portion. As used herein, " the inside " (also referred to as " the inner core " or simply referred to as " the core ") of LNP refers to everything in the outermost lipid layer (such as, the outermost double layer) that separates the lipid nanoparticle from its external environment. It has been observed that along with the further reduction of the ionizable lipid ratio, the size of the hydrophobic core may be reduced and the number of layers is reduced. Unrestrictedly, the LNP of the inorganic particles as described herein is mixed, such as with its maximum encapsulation level, can be substantially free of the hydrophobic core in the inner core or its small region. In these embodiments, the inorganic particles can be at least partially compounded with ionizable lipids. Unrestrictedly, the inorganic particles can be located at the intersection of one or more lipid layers (such as, layer). Advantageously, the aqueous portion in these lipid nanoparticles can accommodate the therapeutic agent and / or diagnostic agent actively loaded with high encapsulation efficiency.

[0030] The lipid nanoparticles typically have an average diameter of 50 to 180 nm, 60 to 150 nm, or 65 to 130 nm, or any range therebetween. The cross-section of the lipid nanoparticles may be elongated or circular.

[0031] Helper lipids

[0032] In the present disclosure, the term "helper lipid" includes any vesicle-forming or liposome-forming lipid. Therefore, helper lipids include amphipathic lipids (e.g., C14-C18 alkyl chains with 0-3 double bonds), wherein the polar (i.e., hydrophilic) region contains phosphate, carboxyl, sulfate, sulfonyl, amino or nitro groups. In some embodiments, the helper lipid is a phospholipid (e.g., phosphatidylcholine, sphingomyelin, etc. or a mixture thereof). Helper lipids can be cationic, anionic or zwitterionic at physiological pH (e.g., pH ~ 7.0), and can be net negatively charged, net positively charged or have 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), tetramyristoyl cardiolipin (14:0), DOTAP (18:1), and in some embodiments, the helper lipid is one or a mixture of two or more of the above. 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 a single helper lipid or a mixture of two or more types of different helper lipids.

[0033] In some embodiments, the helper lipid content is greater than 20mol%, greater than 25mol%, greater than 30mol%, greater than 32mol%, greater than 34mol%, greater than 36mol%, greater than 38mol%, greater than 40mol%, greater than 42mol%, greater than 44mol%, greater than 46mol%, greater than 48mol% or greater than 50mol%. In some embodiments, the upper limit of the helper lipid content is 70mol%, 65mol%, 60mol%, 55mol%, 50mol% or 45mol%. The disclosure also encompasses the sub-range of any combination of the above-mentioned upper and lower numerical limits.

[0034] For example, in certain embodiments, the total helper lipid content is 20 mol % to 70 mol %, or 25 mol % to 70 mol %, or 30 mol % to 70 mol %, or 35 mol % to 70 mol %, or 40 mol % to 70 mol % of the total lipids present in the lipid nanoparticles.

[0035] 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%.

[0036] 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 sub-ranges of any combination of the above upper and lower numerical limits.

[0037] 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 lipids present in the lipid nanoparticles. The phosphatidylcholine lipid content is determined based on the total amount of lipids (including sterols) in the lipid nanoparticles.

[0038] Ionizable lipids

[0039] The term "ionizable lipid" refers to any of a variety of lipid species that carry a net positive or net 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.

[0040] In some embodiments, ionizable lipids include cationic lipids, and in certain embodiments, have a head group comprising an amino group. In selected embodiments, cationic lipids include 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 to 3 double bonds. Non-limiting examples of cationic ionizable lipids include 1,2-dioleoyl-3-dimethylammonium propane (DODAP), 1,2-dioleyloxy-3-dimethylaminopropane (DODMA). These 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-octadecene-3-trimethylammonium propane (DOTMA). In some embodiments, the ionizable lipid is a cationic lipid or a mixture of two or more cationic lipids, for example, two or more of those disclosed herein.

[0041] In some embodiments, the ionizable lipids include anionic lipids. In selected embodiments, the anionic lipids include anionic head groups (e.g., phosphate, carboxyl, sulfate, sulfonyl, nitro, etc.), C14 to C18 alkyl chains, a linker region between the head group and the alkyl chain, and 0 to 3 double bonds, and are optionally PEGylated (PEG is 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 contained in lipid nanoparticles.

[0042] In some embodiments, the ionizable lipid comprises one or more charged lipids as described in WO 2021 / 026647, the entire contents of which are incorporated herein by reference. In some of these embodiments, the charged lipid is a lipid comprising a branched lipid portion L having a structure as shown in Formula I (the definitions of terms from WO 2021 / 026647 are incorporated by reference).

[0043] Chemical formula I:

[0044] A-(V)m -ZL

[0045] (O)

[0046] A is an ionizable, permanently charged, or zwitterionic head group;

[0047] (V) m is optionally -(CR1R2)m-, and m is 1 to 10 or 2 to 6, wherein R1 and R2 are each independently: hydrogen, optionally substituted alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkylalkyl or heterocycle; and

[0048] ZL has the structure shown in the following chemical formula II, IIa or IIb,

[0049] And wherein L is a hydrocarbon structure and has a moiety shown in the following chemical formula IIIc.

[0050] Chemical formula IIIc:

[0051]

[0052] Wherein L1′-L1″-G1-CH-[CH2] q -CH3 represents the scaffold carbon chain of L, and wherein the total number of carbon atoms in the carbon backbone of L is 10 to 30;

[0053] L1′ is a straight hydrocarbon chain containing no heteroatoms and having 5-12 (or more) carbon atoms and 0-3 cis or trans double bonds;

[0054] L1″ is a carbon atom;

[0055] Each X1 is independently selected from ether, ester and carbamate groups;

[0056] L1″′ is the carbon main chain part of the backbone carbon chain L and is composed of G 1 -CH-[CH2] q -CH3 represents, and wherein G 1 is a hydrocarbon chain of 0-4 carbon atoms, optionally having a cis or trans double bond;

[0057] Where n is 0 to 4;

[0058] Where p is 1 to 4;

[0059] Where n+p is 1 to 4;

[0060] q is 0 to 20;

[0061] Wherein S and L1"" are both hydrocarbon side chains and are independently:

[0062] (a) a linear or branched terminated hydrocarbon chain having 0 to 5 cis or trans C=C and 2 to 30 carbon atoms and conjugated to one of the corresponding X1 at any carbon atom in its hydrocarbon chain; or

[0063] (b) a branched hydrocarbon structure represented by chemical formula IIIc,

[0064] wherein each of the L1"" and S hydrocarbon chains in the lipid portion are optionally substituted with heteroatoms, provided that no more than 2 heteroatoms are substituted in the hydrocarbon chain.

[0065] Chemical formula II straight linker structure:

[0066] X1-Lb,

[0067] wherein X1 is optional and X1 is selected from ether, ester and carbamate groups; and

[0068] Lb is a branched lipid represented by chemical formula IIIc.

[0069] Chemical formula IIa branched linker structure:

[0070]

[0071] W is optional;

[0072] W (if present) is a X1 bond, NC(O), NC(O)O or N-OC(O);

[0073] wherein W is optionally substituted with D, which is optionally substituted alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkylalkyl, or heterocycle;

[0074] Each occurrence of (X) n is independently selected - (CR1R2) n -;(X) n n is 0 to 10; and T is optional and is alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkylalkyl or heterocycle, and wherein T is optionally substituted;

[0075] B is a carbon atom connected to L1 and L2 through respective G1 and G2;

[0076] wherein G1 and G2 are independently selected from X1;

[0077] Each of G1 and G2 is connected via an intermediate (G) u The groups are each independently optionally covalently bonded to B as B-(G) u -G1 or B-(G) u -G2;

[0078] Where (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;

[0079] wherein G3 is optional and is selected from X1, and via the intervening (G) u The group is optionally covalently bonded to B as B-(G) u -G3;

[0080] L1 is a branched hydrocarbon represented by chemical formula IIIc;

[0081] L2 is a hydrocarbon chain having 1 to 20 carbon atoms and 0 to 2 cis or trans double bonds, or has a structure shown in chemical formula IIIc;

[0082] L3, if 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 shown in Formula IIIc.

[0083] Chemical formula IIb ring structure:

[0084]

[0085] wherein the curved line represents a ring, E and K show atoms partially forming the structure of the ring, and the ring is a substituted or unsubstituted ring having 3 to 8 ring atoms;

[0086] wherein at least one of L1, L2 and L3 is optionally bonded to a single atom in the ring via respective G1, G2 and G3, wherein each of G1, G2 and G3 is independently optionally bonded via an intervening (G) u Covalently bonded to the corresponding groups in L1, L2 and L3, respectively, as G1-(G) u -L1, G2-(G) u -L2 or G3-(G) u -L3;

[0087] wherein L1 and optionally L2 and / or L3 of chemical formula IIb have the structure of chemical formula IIIc; and

[0088] Wherein part A is optionally selected from one of the following:

[0089] (i) an ionizable cationic moiety selected from:

[0090]

[0091] (ii) a permanently charged portion selected from:

[0092]

[0093] (iii) an ionizable anionic moiety selected from:

[0094]

[0095] (iv) a zwitterionic moiety selected from:

[0096]

[0097] In some embodiments, ZL has a structure as shown in Formula II; wherein L1′ of Formula IIIc has 5 to 9 carbon atoms and has 0 to 2 cis or trans double bonds; wherein G of Formula IIIc 1 is absent, CH2 or CH2CH=CH, and wherein the double bond is cis or trans; wherein L1"" (if present) and S of formula IIIc are independently selected from hydrocarbons having 0-5 cis or trans CH=CH and 2 to 18 carbon atoms; and wherein q is 1 to 9.

[0098] In some embodiments, (v) m Yes (CH2) m , wherein m is 1 to 20; ZL has the structure shown in Formula IIa; wherein W is an ether, ester or carbamate group and D is absent, and (X) n Yes (CH2) n , wherein n is 1 to 10; wherein G1 and G2 are present and are connected via (G) u Covalently bonded to B as B-(G) u -L1 or B-(G) u -L2, where (G) u Yes (CH2) u ; wherein G3-L3 exists and is a hydrocarbon selected from CH3 and CH2CH3; or wherein 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 a structure shown in chemical formula IIIc.

[0099] In some embodiments, ZL has the structure shown in chemical formula IIb, wherein the curve represents a ring and E and K show the atoms that partially form the ring structure, and the ring is a substituted or unsubstituted carbocyclic ring with 3 to 6 ring atoms. In selected embodiments, the ring comprises 3 or 5 carbon atoms. In selected embodiments, at least L1 and L2 are present, and are connected to the ring via respective G1 and G2 groups, and wherein each G1 and G2 group is optionally covalently bonded to the atoms of the ring via intervening (G)u, wherein (G)u is (CH2)u and u is 0 to 10 or 0 to 6.

[0100] In some embodiments, (V) m R1 or R2 is cycloalkyl, which is an optionally substituted monocyclic, bicyclic or tricyclic carbocyclic ring.

[0101] In some embodiments, (V) m R1 and R2 are each independently selected from a heteroatom ring having 4 to 12 ring atoms.

[0102] Ionizable lipid content can be less than 30mol%, less than 25mol%, less than 20mol%, less than 18mol%, less than 15mol%, less than 12mol%, less than 10mol% or less than 5mol%. In certain embodiments, ionizable lipid content is 2mol% to 30mol% of the total lipid present in lipid nanoparticles, or 5mol% to 25mol%, or 7mol% to 20mol%. In some embodiments, ionizable lipid is cationic at physiological pH. In one embodiment, the charge ratio (N / P) of the amine to phosphate of lipid nanoparticles is 3 to 15, 5 to 10, 6 to 9, 8 to 10 or 5 to 8.

[0103] Sterols

[0104] The lipid nanoparticles may optionally include sterols. Examples of sterols include cholesterol or cholesterol derivatives such as cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2′-hydroxyethyl ether, cholesteryl-4′-hydroxybutyl ether, β-sitosterol, fucosterol, etc. In one embodiment, based on the total lipid present in the lipid nanoparticles, the content of sterols is 15mol% to 65mol%, 18mol% to 50mol%, 20mol% to 50mol%, 25mol% to 50mol% or 30mol% to 50mol%. In another embodiment, the sterol is cholesterol, and the content is 15mol% to 65mol%, 18mol% to 50mol%, 20mol% to 50mol%, 25mol% to 50mol% or 30mol% to 50mol% of the total lipid present in the sterol and lipid nanoparticles.

[0105] In one embodiment, the combined (i) sterol content (e.g., cholesterol or a cholesterol derivative thereof); and (ii) auxiliary 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 lipids present in the lipid nanoparticle.

[0106] Hydrophilic polymer-lipid conjugate

[0107] In one embodiment, lipid nanoparticles include a hydrophilic polymer lipid conjugate that can be incorporated into lipid nanoparticles. The conjugate includes a lipid forming a vesicle with a polar head group, and (ii) a hydrophilic polymer chain covalently attached to the head group. The example of a hydrophilic polymer includes polyethylene glycol (PEG) (Nunes et al., 2019, Drug Delivery and Transformation Research (Drug Deliv Transl Res), Vol. 9, No. 1, pp. 123-130), polyvinyl pyrrolidone, polyvinyl methyl ether, polyhydroxypropyl methacrylate, polyhydroxypropyl methacrylamide, polyhydroxyethyl acrylate, polymethacrylamide, polydimethylacrylamide, polymethyl oxazoline, polyethyl oxazoline, polyhydroxyethyl oxazoline, polyhydroxypropyl oxazoline, polysarcosine and polyasparagine. 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.

[0108] The hydrophilic polymer lipid conjugate can account for 0mol% to 5mol%, or 0.5mol% to 3mol%, or 0.5mol% to 2.5mol%, or 0.5mol% to 2.0mol%, or 0.5mol% to 1.8mol% of the total lipid in the nanoparticle. In another embodiment, the PEG-lipid conjugate accounts for 0mol% to 5mol%, or 0.5mol% to 3mol%, or 0.5mol% to 2.5mol%, or 0.5mol% to 2.0mol%, or 0.5mol% to 1.8mol% of the total lipid in the nanoparticle. In certain embodiments, the PEG-lipid conjugate can account for 0mol% to 5mol%, or 0mol% to 3mol%, or 0mol% to 2.5mol%, or 0mol% to 2.0mol%, or 0mol% to 1.8mol% of the total lipid in the nanoparticle.

[0109] Inorganic particles

[0110] The term "inorganic particles" refers to nanometer-sized particles suitable for preparing in lipid nanoparticles as described herein and comprising suitable metals. Inorganic particles have the suitable properties of triggering drug release when applying suitable energy wavelengths. For example, the inorganic particles can be magnetic. In some non-limiting examples, metal is most advantageously biocompatible and nontoxic. Metals include but are not limited to gold, silver, iron, copper, nickel, cobalt, platinum, iridium, alloys of two or more thereof or mixtures thereof. Metals can exist in any form, such as salts (for example, oxides, hydroxides, sulfides, phosphates, fluorides or chlorides) or can be complexed. In some embodiments, metal is gold or silver. In another embodiment, inorganic particles include hybrid gold iron oxides.

[0111] Inorganic particles include, but are not limited to, metal nanoparticles, nanoshells, nanocages, quantum dots, or upconversion nanoparticles. In some embodiments, the inorganic particles are spherical or rod-shaped or nanostar-shaped, most typically spherical.

[0112] Inorganic particles are generally small and have a diameter of less than 20 nm, less than 15 nm, less than 10 nm, or less than 5 nm.

[0113] In the case of metal nanoparticles, the metal can be combined with a ligand, such as a "capping agent". Without limitation, the capping agent can control the growth, aggregation and / or physicochemical properties of the metal nanoparticles. In some embodiments, the capping agent can reduce or block the reactivity around the metal nanoparticles. In some embodiments, the capping agent can act as a reducing agent and a capping agent.

[0114] In some advantageous embodiments, the capping agent makes the metal nanoparticles negatively charged. If the ionizable lipid is a cation at a desired pH (e.g., physiological pH or lower than physiological pH), then the use of a negatively charged capping ligand can contribute to the incorporation of the metal nanoparticles into lipid nanoparticles. In some embodiments, the opposite charge between the ionizable lipid and the metal nanoparticles allows the formation of a combination or complex between negatively charged metal nanoparticles and positively charged lipids, thereby improving encapsulation efficiency. In one embodiment, the capping agent is a macromolecule, such as but not limited to tannic acid or includes a citrate ion.

[0115] In some alternative embodiments, the capping agent can be an amphiphilic molecule comprising a polar head group and a non-polar hydrocarbon tail. Due to the amphiphilic nature of the capping agent, in some embodiments, they provide functionality and / or enhance compatibility with another phase. In a non-limiting example, the non-polar tail interacts with an external medium, while the polar head interacts with the metal atoms of the nanoparticle.

[0116] Loading of reagents of interest

[0117] In some embodiments, LNP comprises a water-soluble loadable reagent of interest present in the aqueous portion of LNP, for example, a therapeutic agent, a diagnostic agent and / or a diagnostic agent. In other embodiments, LNP comprises a hydrophobic or lipophilic loadable reagent of interest present in the lipid portion of LNP, for example, a therapeutic agent, a diagnostic agent and / or a diagnostic agent, or a prodrug thereof. The reagent of interest that can be loaded can be any molecule of interest, for example, a small molecule (for example, a small molecule drug, an imaging agent, etc.), a protein (for example, an antibody, etc.), a peptide, a nucleic acid (for example, a siRNA, etc.).

[0118] In one embodiment, the reagent that is incorporated into the aqueous part of lipid nanoparticle can be actively loaded wherein.The reagent that can use pH gradient load to load comprises one or more ionizable parts, so that the neutral form of described ionizable part allows drug to pass through lipid nanoparticle film, and this part causes reagent to remain encapsulated in liposome to the conversion of charged form.Ionizable part can include amine, carboxylic acid, hydroxyl or any other charged part.The reagent loaded in response to acidic interior can include ionizable part that is charged in response to acidic environment, and the reagent loaded in response to alkaline interior includes part that is charged in response to alkaline environment.In the case of alkaline interior, ionizable part can be used, and it includes but is not limited to carboxylic acid or hydroxyl.In the case of acidic interior, ionizable part can be used, and it includes but is not limited to primary amine, secondary amine and tertiary amine group. In some embodiments, the reagent loaded into the basic interior using pH gradient loading should be a weak acid or have a pKa of 2-6 and a molecular weight of <1500 g / mol, while the reagent loaded into the acidic interior using pH gradient loading should be a weak base or have a pKa of 6-9 and a molecular weight of <1500 g / mol.

[0119] Without limitation, the reagent that pH gradient can load can be antitumor agent, antimicrobial agent or antiviral agent. Non-limiting examples of therapeutic agents that can be loaded into lipid nanoparticles by pH gradient loading method and can therefore 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; antitumor antibiotics, such as mitomycin, bleomycin and dactinomycin; vinca alkaloids, such as vinblastine, vincristine and vinorelbine; 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; cytarabines, such as cytarabine; antimicrobial agents, such as ciprofloxacin and its salts.

[0120] In some embodiments, the loadable agent of interest is a diagnostic agent or an imaging agent (e.g., a contrast agent, such as a radiolabeled agent or MRI contrast agent, a fluorescent probe, etc.). These agents can be incorporated into the aqueous portion of the lipid nanoparticle or the lipid portion of the lipid nanoparticle. For example, but not limited to, a soluble contrast agent can be incorporated into the aqueous portion of the LNP.

[0121] The hydrophobic agent of interest can be readily loaded into the LNP system by simply mixing it with the lipid component (eg, see Example 4).

[0122] Hydrophilic agents can be converted into hydrophobic agents using known methods, and thus converted into lipid loadable, such as the method described in WO / 2020 / 191477, which includes but is not limited to conjugated lipid parts, and the patent is incorporated herein by reference in its entirety. A variety of joints can be used, for example, succinate, ester, amide, hydrazone, ether, carbamate, carbonate, phosphodiester and other conjugated lipid parts. The lipid-conjugated reagent can be a therapeutic agent, a diagnostic agent, a diagnostic agent or any other reagent of interest. The joint between the lipid part and the reagent of interest can be cleaved in vivo (for example, by enzymes, pH, etc.). The lipid-conjugated reagent can be a prodrug, so that it is activated as a therapeutic agent or a diagnostic agent form after release / cleavage of the lipid part, or it can be released as a prodrug and subsequently converted (for example, biochemical conversion) to its active form. Suitable lipids and joints (for example, cleavable and non-cleavable joints) are known, such as described in WO / 2020 / 191477.

[0123] Preparation of nanoparticles

[0124] Hybrid lipid nanoparticles can be prepared using a variety of suitable methods, such as rapid mixing / solvent (e.g., ethanol) dilution. 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, the entire contents of which are incorporated herein by reference.

[0125] In order to incorporate reagent into the aqueous part of lipid nanoparticle, load buffer should be introduced therein to drive the absorption of reagent.There are multiple possible methods that load buffer is introduced into LNP.A possible method is included during the lipid mixing stage with the solvent (for example, ethanol), and load buffer is mixed in the aqueous medium containing inorganic particles.However, the aqueous dispersion of colloidal metal (for example gold) may be sensitive to ionic strength.In some embodiments, it is possible that the precipitation of LNP can occur when buffer is introduced during the mixing stage.In order to solve the possibility of this LNP precipitation when introducing load solution during mixing, load solution can be added to the LNP comprising the metal of encapsulation after LNP is formed, and then load solution is absorbed in the lipid nanoparticle.

[0126] In these embodiments, the method comprises that inorganic particles are embedded in lipid nanoparticles to produce the lipid nanoparticles comprising the inorganic particles (for example, metal nanoparticles) embedded. This relates to merging two separate streams, the first preparation of lipids dissolved in a solvent (for example, ethanol or other suitable solvents) and the second preparation of the aqueous solution of inorganic particles. Two streams are merged in a suitable mixing device to produce the stream merged, thereby forming the lipid nanoparticles of the embedded inorganic particles. Subsequently, loading buffer is added in the external solution of the lipid nanoparticles thus formed. Because the external solution comprises a solvent (for example, ethanol or other suitable solvents) for forming the LNP comprising inorganic particles, and this solvent can contribute to the incorporation of loading buffer to the aqueous part of lipid nanoparticles by enhancing the permeability of the LNP lipid layer, this makes loading buffer embedded in lipid nanoparticles, thereby producing the lipid nanoparticles comprising inorganic particles and the loading buffer embedded in it or in the compartment. In order to produce a transmembrane chemical gradient, the original external medium of lipid nanoparticles is replaced with a new external medium with a material (for example, proton) having different concentrations of the driving load. The method then includes introducing an actively loadable agent into the external solution of the lipid nanoparticles comprising an encapsulated loading buffer and actively loading the agent into the lipid nanoparticles, thereby producing lipid nanoparticles that encapsulate both the inorganic particles and the actively loadable agent.

[0127] The replacement of external medium can be accomplished by multiple techniques, such as by passing lipid nanoparticles through a gel filtration column, for example, a Sephadex column, which has been balanced with a new medium (as described in the following examples), or by centrifugation, dialysis or related techniques. For pH gradient loading, the internal medium can be acidic or alkaline relative to the external medium. In these embodiments, after setting up the pH gradient, the reagent that the pH gradient can load is added to the mixture, and the reagent is encapsulated in the lipid nanoparticles as described above.

[0128] Loading using a pH gradient can be performed 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.

[0129] Although pH gradient loading is described, active loading includes the use of any suitable transmembrane chemical gradient across the LNP membrane to induce absorption of the active loadable agent after LNP formation. This may include a gradient of one or more ions including Na + , K + , H +and / or protonated nitrogen moieties. 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 via a drug-binding agent.

[0130] Nanoparticle morphology

[0131] The lipid nanoparticle comprises a core that encapsulates both inorganic particles and an agent, and in some embodiments, the agent is a therapeutic agent or an imaging agent. The term "core" or "inner core" refers to an embedding or at least partially enclosed volume of a lipid nanoparticle comprising 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 a quarter of the core contains an aqueous portion, or at least about a third of the core contains an aqueous portion, or at least about half of the core contains an aqueous portion, as determined qualitatively by cryo-EM or other applicable techniques. In one embodiment, the shape of the lipid nanoparticle is round or elongated in cross section.

[0132] This unique morphology may depend on the ratio of "double layer" lipids (helper lipids) in lipid nanoparticles. It has been observed that when helper lipids (e.g., DSPC) increase, helper lipids first form a monolayer around the hydrophobic core region, and then form a double layer surrounding the core. Along with the further reduction of the ionizable lipid ratio, the size of the hydrophobic region decreases and the number of layers increases. In a non-limiting manner, it can be proposed that LNPs containing negatively charged inorganic particles represent such a limiting case under the maximum inorganic particle (e.g., gold nanoparticles (GNPs)) embedding level: there is basically no hydrophobic region in the core, and the inorganic particles and ionizable lipids meet or combine and are located at the intersection of the layers.

[0133] In one embodiment, as determined by measuring the zeta potential of LNP as described herein, the lipid nanoparticle surface is substantially uncharged. This may be due to the outer lipid layer (e.g., double layer) having a low level of ionizable lipid and high auxiliary lipid content. In alternative embodiments, LNP is single layer or multilayer.

[0134] Inorganic particles can be combined or complexed with ionizable lipids. In some embodiments, the inorganic particles of encapsulation are present in the lipid nanoparticles in the particle region, wherein two lipid layers contact, as detected by cryo-TEM microscopy. However, it should be understood that the present invention is not limited to the inorganic particles in the lipid nanoparticles incorporating position or property. That is to say, the term "encapsulated" is not intended to be limited to any specific interaction between inorganic particles and lipid nanoparticles. Inorganic particles can be incorporated into the aqueous portion, incorporated into any lipid layer, or incorporated into both.

[0135] Pharmaceutical preparations

[0136] In some embodiments, the disclosure provides a method for treating a cell in a subject or imaging the cell by administering at least one lipid nanoparticle to a cell in vivo. The method for processing or imaging cells may further include using an external energy source, such as a light source, a laser (continuous wave (cw) or pulse), an X-ray or a gamma ray. The energy source will cause at least part of the lipid nanoparticle content to be released to achieve imaging and / or therapeutic effect. As known to those skilled in the art, irradiating the lipid nanoparticle with a suitable energy source increases the degradation rate of the lipid nanoparticle.

[0137] In some embodiments, the lipid nanoparticles of the present disclosure are part of a pharmaceutical composition and are administered to treat and / or prevent disease conditions. Treatment can provide prophylactic, preventive, ameliorative or therapeutic benefits. The pharmaceutical composition will be administered in any suitable dosage. In one embodiment, the pharmaceutical composition is administered parenterally (i.e., intra-arterial, intravenous, subcutaneous or intramuscular). In another embodiment, the pharmaceutical composition is used for intratumoral or intrauterine administration. In another embodiment, the pharmaceutical composition is administered intranasally, intravitreally, subretinaly, intrathecally or by other topical routes.

[0138] The pharmaceutical composition can also include one or more pharmaceutically available excipients. Excipients are substances contained in pharmaceutical compositions, the purpose of which is long-term stability, filling of solid preparations containing a small amount of effective active ingredients (i.e., can play the role of "volume expander", "filler" or "diluent"), or in some cases, enhancing the delivery of active ingredients in the final dosage form, such as helping drug absorption, reducing viscosity or changing (usually increasing) solubility. Excipients can also play a role in promoting / improving production, for example, as anti-adhesives, bonding agents, coatings, glidants, lubricants, preservatives, adsorbents and / or vehicles (for liquid and gel preparations). The term excipient includes the terms "carrier" and "diluent". Non-limiting examples of suitable excipients include any suitable buffer, stabilizer, salt, antioxidant, complexing agent, tonicity agent, cryoprotectant, lyophilization protective agent, suspending agent, emulsifier, antimicrobial agent, preservative, chelating agent, bonding agent, surfactant, wetting agent, non-aqueous vehicle such as fixed oils or polymers for sustained release 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 edition (Gennaro et al., ed., Lippincott Williams & Wilkins Philadelphia), each of which is incorporated by reference in its entirety. As used herein, "pharmaceutically acceptable" refers to a substance that is acceptable for use in pharmaceutical applications from a toxicological point of view and that does not adversely interact with the active ingredient. Thus, pharmaceutically acceptable carriers are those that are compatible with the other ingredients in the formulation and are biologically acceptable. Supplementary active ingredients may also be introduced into the pharmaceutical composition. Excipients include, but are not limited to, binders, fillers, flow aids / glidants, disintegrants, lubricants, stabilizers, surfactants, and the like.

[0139] 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 improving symptoms, reducing disease progression, improving prognosis and reducing recurrence.

[0140] In some embodiments, lipid nanoparticles have long circulation life, which may be conducive to entering extrahepatic tissue. In some embodiments, lipid nanoparticles have improved expansibility. In some embodiments, lipid nanoparticles improve the internalization of GNP (or other metal nanoparticles) to avoid immune response problems. In some embodiments, lipid nanoparticles have the ability of improved effective encapsulation of drug goods.

[0141] Triggered release of loaded reagent of interest

[0142] Lipid nanoparticles can be designed to accumulate in the tumor microenvironment, where they only release the encapsulated therapeutic agent at the target cell (such as cancer cell). In addition, the vascular permeability (≤200nm) of lipid nanoparticles (described vascular permeability is increased by the enhanced permeability and retention (EPR) effect known to increase) provides enhanced 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).

[0143] 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 in the tumor microenvironment by stimulus-activated methods. Such internal stimuli-responsive delivery systems are advantageous because they can destabilize lipid nanoparticles (i.e., by degrading their structural components ( 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)).

[0144] Compared to internally stimulated release systems, externally stimulated responsive systems (e.g., using magnetic, ultrasonic, thermal, microwave, radiofrequency, or light stimulation) have the potential to release their encapsulated cargo in the target microenvironment independently of differences in cellular mechanisms / phenomena between target and non-target sites (Chander et al., 2021, Small, Vol. 17, No. 21, pp. 2008198; Mathiyazhakan et al., 2014, Colloids and surfaces. B, Biointerfaces, Vol. 126, pp. 569-574). Examples include: mild localized hyperthermia-triggered processes (e.g., ThermoDox TM technology), as well as phototherapy and photodynamic therapy.

[0145] Laser-triggered drug release from lipid nanoparticles can ensure a sharp transition temperature change, i.e., by focusing the laser beam only on the target tissue (deep in the body) without damaging surrounding non-cancerous cells. This form of light-triggered drug release utilizes a pulsed laser (e.g., with femtosecond, picosecond, or nanosecond pulses) with a peak in the near-infrared (NIR) region, and therefore can deliver stronger and more focused energy (e.g., delivering a highly localized temperature increase of a few nanoseconds within a few nanometers without excessive tissue heating) to specifically release cargo compared to other light-triggered systems. To achieve this effect, the formulation of lipid nanoparticles is adjusted to incorporate plasmon nanoparticles inside them. This enables the amplified, localized electromagnetic plasma fields to be specifically focused inside the liposome target to induce 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 aplasmonic 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). Due to the strong and sudden response of plasmonic nanoparticles to incident light, on / off resonant irradiation with optimized laser energy density can promote controlled local energy absorption and release of energy, whether it is a single irradiation or multiple irradiations (for example, by adjusting the spatial overlap between pulses and exposure time), thereby enabling site-specific drug release in the tumor microenvironment without damaging non-target cells (Pustovalov, 2005, Chemical Physics, Vol. 308, pp. 103-108; Pustovalov et al., Laser Physics Letters, Vol. 5, pp. 775-792).

[0146] LNP disclosed herein can be triggered by a variety of stimuli to release the encapsulated loaded reagents therein, which provides a more targeted release of the loaded reagents, thereby leading to increased efficacy and / or reduced side effects. Common stimuli include, but are not limited to, light (electromagnetic radiation), magnetic fields, temperature, ultrasound, pH, redox or biochemical stimulation (e.g., enzymes, etc.).

[0147] In one aspect, the LNPs or compositions disclosed herein are administered to a subject, and the microenvironment of the target tissue (eg, a tumor) triggers the release of the loaded agent (eg, an anticancer agent, a diagnostic agent, or a tumor imaging agent).

[0148] In another aspect, LNP or composition disclosed herein are applied to a subject, and an external stimulus (e.g., at a target site of the body) is applied to release the loaded reagent. Specific non-limiting examples of external stimulus triggering release are provided in Examples 6-8. For example, but not limited to, visible light / infrared light can be used to trigger the release of an agent of interest from a gold-containing or silver-containing LNP, radio frequency irradiation can be used to trigger the release of an agent of interest from a gold-containing LNP, and a magnetic field can be used to trigger the release of an agent of interest from an LNP containing iron oxide, cobalt or nickel, or an alloy of gold and any of the above-mentioned magnetic metals. In some embodiments, the inorganic particles include iron oxide, and the external stimulus is a magnetic field. In some embodiments, the inorganic particles include gold, and the external stimulus is electromagnetic irradiation (e.g., NIR or UV). In some embodiments, the inorganic particles include gold or silver, and the external stimulus is irradiation from a light source or by a laser. In some embodiments, the inorganic particles are metal nanoparticles (e.g., gold or silver), and the stimulation is irradiation (optionally using a laser) at a wavelength that is resonant or non-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 a pulse width of microseconds, nanoseconds, picoseconds, or femtoseconds. In some embodiments, the irradiation is a femtosecond laser with a wavelength that is resonant or non-resonant with the metal nanoparticles, wherein the metal nanoparticles are plasmons.

[0149] A series of non-limiting embodiments are defined below.

[0150] Embodiment 1. A lipid nanoparticle comprising:

[0151] A helper lipid and an ionizable lipid, wherein the ionizable lipid is present in an amount of 2 mol % to 30 mol % relative to the total lipids;

[0152] at least one lipid layer surrounding an inner core, the inner core having at least one aqueous portion;

[0153] encapsulated inorganic particles; and

[0154] The agent of interest, wherein the agent of interest is a hydrophilic agent present in the at least one aqueous portion or a lipophilic agent present in the at least one lipid layer.

[0155] Embodiment 2. The lipid nanoparticle of embodiment 1, wherein the agent of interest is hydrophilic and is present in the at least one aqueous portion.

[0156] Embodiment 3. The lipid nanoparticle of embodiment 2, wherein the agent of interest is precipitated in at least one aqueous portion.

[0157] Embodiment 4. A lipid nanoparticle according to embodiment 2 or 3, wherein at least one aqueous portion is acidic and wherein the agent of interest is a weak base.

[0158] Embodiment 5. A lipid nanoparticle according to embodiment 2 or 3, wherein at least one aqueous portion is basic and wherein the agent of interest is a weak acid.

[0159] Embodiment 6. The lipid nanoparticle of embodiment 1, wherein the agent of interest is lipophilic and present in the at least one lipid layer.

[0160] Embodiment 7. The lipid nanoparticle of embodiment 1, wherein the agent of interest is a hydrophilic agent conjugated to the lipid portion via a cleavable linker and is present in the at least one lipid layer.

[0161] Embodiment 8. The lipid nanoparticle according to any one of embodiments 1 to 7, wherein the inorganic particles present in the core are metal nanoparticles.

[0162] Embodiment 9. The lipid nanoparticle according to any one of embodiments 1 to 8, wherein the inorganic particles comprise gold or iron.

[0163] Embodiment 10. The lipid nanoparticle of any one of embodiments 1 to 9, wherein the inorganic particles present in the core are colloids.

[0164] Embodiment 11. The lipid nanoparticle of any one of embodiments 1 to 10, wherein the inorganic particles present in the core have a diameter of 1 to 20 nm.

[0165] Embodiment 12. The lipid nanoparticle of any one of embodiments 1 to 11, wherein the lipid nanoparticle has an average diameter of 50 to 200 nm.

[0166] Embodiment 13. The lipid nanoparticle of claim 12, wherein the lipid nanoparticle has an average diameter of 70 to 150 nm.

[0167] Embodiment 14. A lipid nanoparticle according to any one of embodiments 1 to 13, wherein the at least one lipid layer is bilayer (two-layer) or multilayer.

[0168] Embodiment 15. The lipid nanoparticle of embodiment 14, wherein the auxiliary lipid is a neutral lipid, and wherein the lipid component of the nanoparticle further comprises at least one of cholesterol and a hydrophilic polymer-lipid conjugate.

[0169] Embodiment 16. The lipid nanoparticle according to any one of embodiments 1 to 15, wherein the helper lipid is present at a concentration of at least 20 mol %, optionally at least 30 mol % or optionally 40 mol %.

[0170] Embodiment 17. The lipid nanoparticle of any one of embodiments 1 to 16, wherein the ionizable lipid is present at 5 mol % to 15 mol % relative to the total lipids.

[0171] Embodiment 18. The lipid nanoparticle of any one of embodiments 1 to 17, wherein a pH gradient is present on the lipid layer.

[0172] Embodiment 19. The lipid nanoparticle of any one of embodiments 1 to 18, wherein the inorganic particle is negatively charged.

[0173] Embodiment 20. The lipid nanoparticle of any one of embodiments 1 to 19, wherein the inorganic particle comprises a negatively charged cap.

[0174] Embodiment 21. The lipid nanoparticle of any one of embodiments 1 to 20, wherein the ionizable lipid is a cationic at physiological pH.

[0175] Embodiment 22. A lipid nanoparticle according to any one of embodiments 1 to 21, wherein the inorganic particle is complexed with the ionizable lipid and the inorganic particle is located at the intersection of the layers of the lipid layer.

[0176] Embodiment 23. A method for producing lipid nanoparticles encapsulating an inorganic particle and an agent of interest, the method comprising:

[0177] (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 the inorganic particles in the combined stream;

[0178] (ii) introducing a loading medium into the external solution of the lipid nanoparticle thus formed, the external solution comprising the solvent, and causing the loading medium to be entrapped in the lipid nanoparticle, thereby producing a lipid nanoparticle comprising the entrapped loading medium and the inorganic particle in its internal compartment; and

[0179] (iii) introducing an agent of interest into an external solution of the lipid nanoparticles comprising an embedded loading buffer, and allowing the agent of interest to be actively loaded into the lipid nanoparticles in response to the embedded loading medium, thereby generating lipid nanoparticles encapsulating inorganic particles and the agent of interest.

[0180] Embodiment 24. The method according to embodiment 23, wherein the pH of the aqueous solution of the inorganic particles in step (i) is less than 5.5.

[0181] Embodiment 25. A method according to embodiment 23 or 24, wherein the lipid dissolved in the first formulation comprises an ionizable lipid, optionally a cationic lipid.

[0182] Embodiment 26. A method according to 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, thereby making the ionizable cationic lipid charged.

[0183] Embodiment 27. The method of any one of embodiments 23 to 26, wherein the first and second formulations are pumped and mixed in a "T" type junction mixer.

[0184] Embodiment 28. A method according to any one of embodiments 23 to 27, wherein the lipid nanoparticle external solution is exchanged with a solution having a pH at least one pH unit higher than the pH of the loading buffer.

[0185] Embodiment 29. A method according to embodiment 28, wherein the loading medium is added to the lipid nanoparticles before exchanging the external solution.

[0186] Embodiment 30. The method of any one of embodiments 23 to 29, wherein the solvent in the first formulation is ethanol.

[0187] Embodiment 31. A method according to any one of embodiments 23 to 30, wherein the lipids in the first preparation comprise auxiliary lipids and ionizable lipids, wherein the ionizable lipids are present at 2 mol % to 30 mol %, optionally at 5 mol % to 15 mol % relative to the total lipids.

[0188] Embodiment 32. A method according to 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%.

[0189] Embodiment 33. A method for producing lipid nanoparticles comprising an inorganic particle core and a lipophilic agent of interest, the method comprising:

[0190] 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 the inorganic particle cores in the combined stream;

[0191] wherein the lipids in the first formulation comprise a helper lipid, an ionizable lipid, and a lipophilic agent of interest;

[0192] wherein the ionizable lipid is present in an amount of 2 mol % to 30 mol % relative to the total lipids, and optionally in an amount of 5 mol % to 15 mol % relative to the total lipids; and

[0193] Wherein, the helper lipid is present in a concentration of at least 20 mol %, optionally at least 30 mol %, optionally at least 40 mol %.

[0194] Embodiment 34. A method according to embodiment 33, wherein the agent of interest is a hydrophilic agent of interest conjugated to a lipid portion.

[0195] Embodiment 35. A method according to embodiment 34, wherein the lipid portion is conjugated to the hydrophilic agent of interest via a cleavable linker.

[0196] Embodiment 36. A 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.

[0197] Embodiment 36. Use of a lipid nanoparticle according to any one of embodiments 1 to 22 for delivering the agent of interest to a subject, wherein the lipid nanoparticle is used for administration to the subject, followed by administration of a stimulus to an area of ​​the subject, wherein the stimulus causes the inorganic particles in the lipid nanoparticle to cause the agent of interest to be released from the lipid nanoparticle, optionally wherein the stimulus is electromagnetic radiation, optionally from a light source or laser.

[0198] Embodiment 37. The use according to embodiment 36, wherein the agent of interest is a therapeutic agent for treating a disease or condition in the subject.

[0199] Embodiment 38. The use according to embodiment 36, wherein the agent of interest is an imaging agent, and wherein the use further comprises imaging the area of ​​the subject.

[0200] Embodiment 39. The use according to embodiment 36, wherein the agent of interest is a prodrug comprising a lipophilic therapeutic agent conjugated to a lipid portion via a cleavable linker, wherein the use further comprises causing cleavage of the cleavable linker, and after the cleavage, the therapeutic agent treats the disease or condition of the subject.

[0201] Embodiment 40. A lipid nanoparticle comprising:

[0202] 2 mol% to 30 mol% ionizable lipid content;

[0203] at least one of a hydrophilic polymer-lipid conjugate and a sterol;

[0204] A helper lipid content greater than 30 mol% to form a bilayer surrounding the aqueous portion;

[0205] Inorganic particles present in the bilayer; and

[0206] a therapeutic agent and / or an imaging agent present in said aqueous portion,

[0207] Wherein, the therapeutic agent and / or imaging agent is releasable from the lipid nanoparticles by irradiation.

[0208] Embodiment 41. A lipid nanoparticle according to embodiment 40, wherein the therapeutic agent or imaging agent is precipitated in the aqueous portion.

[0209] Embodiment 42. The lipid nanoparticle of embodiment 40 or 41, wherein the inorganic particles are metal nanoparticles and comprise gold or iron.

[0210] Embodiment 43. The lipid nanoparticle of embodiment 42, wherein the metal nanoparticles present in the aqueous portion are colloids.

[0211] Embodiment 44. The lipid nanoparticle of any one of embodiments 40 to 43, wherein the lipid nanoparticle has an average diameter of 50 to 200 nm.

[0212] Embodiment 45. The lipid nanoparticles of embodiment 44, wherein the lipid nanoparticles have an average diameter of 70 to 150 nm.

[0213] Embodiment 46. The lipid nanoparticle of embodiment 45, wherein the metal nanoparticles present in the bilayer have a diameter of 1 to 20 nm.

[0214] Embodiment 47. The lipid nanoparticle of any one of embodiments 40 to 46, wherein the auxiliary lipid is a phosphatidylcholine lipid.

[0215] Embodiment 48. The lipid nanoparticle of any one of embodiments 40 to 47, wherein the helper lipid is present at a concentration of at least 35 mol %.

[0216] Embodiment 49. The lipid nanoparticle of any one of embodiments 40 to 47, wherein the helper lipid is present at a concentration of at least 40 mol %.

[0217] Embodiment 50. The lipid nanoparticle of any one of embodiments 40 to 47, wherein the helper lipid is present at a concentration of at least 45 mol%.

[0218] Embodiment 51. The lipid nanoparticle of any one of embodiments 40 to 50, wherein the cationic lipid is present at 5 mol% to 15 mol% relative to the total lipids.

[0219] Embodiment 52. The lipid nanoparticle of any one of embodiments 40 to 51, wherein a pH gradient is present on the lipid layer.

[0220] Embodiment 53. A lipid nanoparticle according to embodiment 52, wherein the aqueous core is acidic and the solution outside the lipid nanoparticle is basic.

[0221] Embodiment 54. The lipid nanoparticle of any one of embodiments 40 to 53, wherein the therapeutic agent is a weak base and is pH gradient loadable.

[0222] Embodiment 55. The lipid nanoparticle of any one of embodiments 40 to 54, wherein the metal nanoparticle is negatively charged.

[0223] Embodiment 56. The lipid nanoparticle of any one of embodiments 40 to 55, wherein the metal nanoparticle comprises a negatively charged cap.

[0224] Embodiment 57. The lipid nanoparticle of any one of embodiments 40 to 56, wherein the ionizable lipid is a cationic below physiological pH.

[0225] Embodiment 58. A method for producing lipid nanoparticles according to any one of embodiments 1 to 22 or 40 to 57, the method comprising an ethanol mixing method.

[0226] Embodiment 59. Use of a lipid nanoparticle according to any one of embodiments 1 to 22 or 40 to 57 for treating a mammalian subject in need of treatment, comprising triggered release of an agent at a target site in the body.

[0227] 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 laser.

[0228] Embodiment 61. The use of lipid nanoparticles according to embodiment 60, wherein the irradiation has a wavelength that is resonant or non-resonant with the plasmon peak of the metal nanoparticles.

[0229] Embodiment 62. The use of lipid nanoparticles according to embodiment 61, wherein the triggered release is caused by laser and is resonant.

[0230] Embodiment 63. The use of lipid nanoparticles according to embodiment 61, wherein the laser is continuous wave or pulsed.

[0231] Embodiment 64. The use of lipid nanoparticles according to embodiment 61, wherein the laser is pulsed with a pulse width of microseconds, nanoseconds, picoseconds or femtoseconds.

[0232] Embodiment 65. The use of lipid nanoparticles according to embodiment 61, wherein the irradiation is a femtosecond laser having a wavelength that is resonant or non-resonant with the metal nanoparticles, wherein the metal nanoparticles are plasmons.

[0233] Embodiment 66. A method of medical treatment comprising administering a lipid nanoparticle according to any one of embodiments 1 to 22 or 40 to 57 to a mammalian subject in need of such treatment; and subjecting the lipid nanoparticle to irradiation to trigger release of the agent at a target site in the body.

[0234] Embodiment 67. A method according to embodiment 66, wherein the release is caused by irradiation from a light source or by laser.

[0235] Embodiment 68. The method of embodiment 66, wherein the irradiation has a wavelength that is resonant or non-resonant with the plasmon peak of the metal nanoparticles.

[0236] Embodiment 69. A method according to embodiment 66, wherein the triggered release is caused by laser and is resonant.

[0237] Embodiment 70. A method according to embodiment 66, wherein the laser is continuous wave or pulsed.

[0238] Embodiment 71. A method according to embodiment 66, wherein the laser is pulsed with a pulse width of microseconds, nanoseconds, picoseconds, or femtoseconds.

[0239] Embodiment 72. The method of embodiment 66, wherein the irradiation is a femtosecond laser having a wavelength that is resonant or non-resonant with the metal nanoparticles, and wherein the metal nanoparticles are plasmons.

[0240] The following examples are intended to illustrate the preparation and properties of specific lipid nanoparticle formulations, but are in no way intended to limit the scope of the present invention.

[0241] Example

[0242] Material

[0243] 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, Alabama). Cholesterol (Chol), sodium acetate, ammonium sulfate (AS), and doxorubicin hydrochloride were obtained from Sigma-Aldrich Canada Ltd. (Oakville, Ontario, Canada). Phosphate-buffered saline was from GIBCO (Carlsbad, California). Dialysis membranes (molecular weight cutoff 12000-14000 Da) were from Spectrum Laboratories, Coolmongo, California. Amicon Ultracel centrifugal units (10 kDa MWCO) were from Millipore (Billerica, Massachusetts). Tannic acid-stabilized, negatively charged, monodisperse spherical gold nanoparticles (5 nm diameter, particle concentration 5.5 × 10 13 Anion exchange spin columns (Vivapure DMini H) were obtained from SartoriusStedim Biotech, Aubagne, France. QuantiFluo TMThe fluorescent ammonia assay kit was obtained from BioAssay Systems (Hayward, CA). The Cholesterol E Total Cholesterol Assay Kit was provided by Wako Diagnostics (Richmond, VA).

[0244] method

[0245] Synthesis of gold / lipid hybrid nanoparticle systems.

[0246] LNP-GNPs were prepared by a variation of the ethanol mixing method using a T-tube mixer (Hirota et al., Biotechniques, 1999, 27(2), 286-90; the document is incorporated herein by reference). Briefly, lipid nanoparticles-gold nanoparticles (LNP-GNPs) were prepared by mixing an appropriate volume of lipid stock solution in ethanol buffer with an aqueous phase containing gold nanoparticles (GNPs) using a T-tube mixer. Lipids (DODAP, DSPC, Chol and PEG-DSPE) were dissolved in 100% ethanol at a molar ratio of 10 / 49 / 40 / 1 to a final lipid concentration of 10 mg / ml. Aqueous gold colloids (nominal particle size of 5 nm, particle concentration of 5.5 mg / ml, as provided by the manufacturer) were concentrated using a centrifugal concentrator, and an appropriate volume of the concentrated dispersion was then dissolved in 25 mM sodium acetate buffer (pH 4.0) to achieve the desired GNP number density level. The aqueous medium must be acidified to fully protonate (positively charged) the cationic lipids to facilitate binding to the negatively charged tannic acid cap of the GNPs. Lipids dissolved in ethanol and GNP aqueous dispersions were pumped by two syringe pumps (volume flow ratio of 3:1 (water to ethanol) at pump rates of 15 and 5 ml / min, respectively) and mixed in a "T" manifold where two syringes containing organic and aqueous streams were connected to a manifold connector (1 / 16", 0.02 inch (in) through hole, IDEX Health & Science Part # P-712). Lipids were added at different gold / lipid (Au / L) ratios (1.1×10 13 to 8.8×10 13Particles / μmol lipid) are combined with GNPs. After mixing, the gold / lipid dispersion is dialyzed with formulation buffer to remove ethanol, or dialyzed with 1× phosphate buffered saline (pH 7.4) to remove residual ethanol and neutralize the buffer. After dialysis is complete, the average diameter size of spontaneously formed LNP-GNPs and ζ-potential (Zetasizer Nano ZS, Malvern Instruments Inc., Westborough, Massachusetts) are determined. The lipid concentration is determined by measuring total cholesterol using an enzyme assay. The absorbance at 520nm (maximum absorbance of 5nm spherical GNPs) in the samples collected before and after removing unembedded gold using anion exchange centrifugal columns is measured and the respective Au / L ratios are compared to quantify colloidal gold, thereby measuring the GNP embedding efficiency. The absorbance measurement is performed by 1% TritonX-100 cracking LNP-GNPs and releasing the embedded gold nanoparticles.

[0247] Synthesis of a gold / lipid nanoparticle system with remotely loadable GNPs.

[0248] In order to achieve a LNP-GNP system capable of remote loading and stable retention of drug cargo, a freshly prepared lipid / gold mixture (prepared as described above) dispersed in an acetate buffer containing 25% ethanol was doped with a concentrated solution of ammonium sulfate (AS). In brief, 1 ml of an AS aqueous solution (usually 0.9, 1.35, and 1.8 M) was added dropwise to a 2 ml vortexed lipid / gold dispersion, and the resulting mixture was then placed in a dialysis bag and dialyzed with phosphate buffered saline to remove ethanol, unembedded AS, and the pH was raised to 7.4. The LNP-GNPs produced by this procedure were embedded with AS in sufficient amounts to provide absorption and stable retention of externally added drugs through an active loading mechanism 27. The AS embedding percentage was determined by measuring the concentration of ammonium in the samples collected before and after dialysis (i.e., before and after removing unembedded ammonium) using a fluorescent ammonia / ammonium assay kit. Measurements were performed in the presence of 1% TritonX-100 to crack the LNP-GNPs and release their contents. Particle size and lipid concentration measurements were performed as described above.

[0249] Loading of therapeutic agents into LNP-GNPs.

[0250] Before loading, the LNP-GNP system containing ammonium sulfate was concentrated to about 10mg / ml lipid using a centrifugal concentrator. Doxorubicin hydrochloride was dissolved in saline with 5mg / ml, and mixed with LNP-GNP dispersion to provide desired drug / lipid (D / L) ratio. Then, the sample was cultivated at 60°C to provide optimal loading conditions. Before detecting embedded drugs, unembedded doxorubicin was removed by running samples on Sephadex G-50 centrifugal columns. Doxorubicin was measured by fluorescence intensity (excitation and emission wavelengths were 480 and 590nm, respectively) with Perkin-Elmer LS50 fluorometer (Perkin-Elmer, Norwalk, Connecticut), and the value of 100% release was obtained by adding isopropanol to a final concentration of 50% volume. Drug and lipid levels were quantified in samples obtained before and after separation of unentrapped drug from LNP-GNP encapsulated drug by size exclusion chromatography using Sephadex G-50 spin columns, and the respective drug / lipid ratios were compared to determine drug loading efficiency.

[0251] Cryo-transmission electron microscopy (Cryo-TEM) analysis of LNP morphology.

[0252] The hybrid LNP-GNPS system was imaged using a FEI Tecnai G20 TEM (FEI, Hillsboro, OR), as described by Leung et al. in J Phys Chem B, 2015, 119 (28), 8698-706 and Witzigmann et al. in Adv Drug Deliv Rev, 2020, 159, 344-363, which are incorporated herein by reference. Before imaging, the sample was concentrated to about 20 mg / mL total lipids, and 3-5 μL aliquots of the concentrated dispersion were transferred to a glow-discharged copper grid in a FEI Mark Iv Vitrobot. The sample was then flash-frozen in liquid ethane to produce vitreous ice. The frozen sample was stored in liquid nitrogen until imaging. The TEM was operated at 200 kV in low-dose mode and images were acquired at a nominal weak focus of 2–4 μm using a bottom-mounted FEI high-resolution CCD camera (FEI, Hillsboro, OR). Sample preparation and image acquisition were performed at the UBC Bioimaging Facility (Vancouver, BC).

[0253] Measuring hydrophobic prodrugs in LNP-GNPs.

[0254] Using a photodiode array detector (PDA) Acquity TMUPLC system, quantification of INT-D034 by ultra-high pressure liquid chromatography (UPLC); using Empower TM Data acquisition software version 3.0 (Waters, USA) was used. Acquity TM BEH C18 column (1.7 μm, 2.1 × 100 mm) was used for separation 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). The method was run at a column temperature of 55 °C for 6 minutes and the analytes were measured by monitoring the PDA detector at a wavelength of 239 nm.

[0255] Example 1. LNPs containing high levels of helper lipids can be produced, containing inorganic particles as well as an aqueous portion

[0256] This embodiment illustrates that LNP containing inorganic particles can be prepared by solvent mixing method, and the inside of the inorganic particles has an aqueous part. When the ionizable lipid content is lower than the content used in the conventional preparation of nucleic acid in LNP and the auxiliary lipid content is higher than the content used in the conventional preparation, this LNP morphology is observed. Previous work mainly uses the lipid composition of ionizable lipid / cholesterol / DSPC / PEG-lipid with a molar ratio of 50 / 38.5 / 10 / 1.5 to encapsulate nucleic acid.

[0257] Select the lipid composition with 10mol% ionizable lipid (DODAP / DSPC / cholesterol / PEG-lipid, 10 / 49 / 40 / 1, mol / mol) to confirm inorganic particles (gold nanoparticles (GNPs)) loading. Use T-tube mixer to quickly mix the lipid mixture dissolved in ethanol with the aqueous solution containing negatively charged inorganic particles (GNPs). The aqueous solution is buffered at pH 4, so that the ionizable lipid is protonated and therefore positively charged. After the mixing step, ethanol is removed by dialysis and the external medium is replaced by the phosphate buffered saline of pH 7.4, except for the micrographs shown in Figures A and C (wherein only ethanol is removed, and by dialysis with 25mM sodium acetate buffer, the sample is maintained at pH 4).

[0258] like Figure 1 As shown in A, at pH 4 (after removal of ethanol by dialysis), the LNPs formed in the absence of GNPs were small (~60 nm) unilamellar vesicles, some of which were observed to be in a "hemifused" state. In contrast, adjusting the pH to 7.4 resulted in the production of "fish-eye" structures with an internal lipid core ( Figure 1B). Such structures are formed due to high "helper" lipid content and can be accurately modeled under the following assumption: the inner inner lipid core region corresponds to an oil core comprising ionizable cationic lipids in neutral form, which is surrounded by a monolayer of helper lipids and PEG lipids. On the other hand, the surrounding bilayer can consist of or essentially consist of the helper lipids and PEG-lipids that form the bilayer.

[0259] When GNPs are present in formulation buffer, the particles formed at pH 4 are unilamellar or bilamellar vesicles with a diameter of ∼100 nm. GNPs are either located within a bilamellar vesicle or at the interface between two unilamellar vesicles to produce a “dumbbell” structure ( Figure 1 C, indicated by arrows). After the pH was raised to pH 7.4, the observed particles were of comparable size but were almost exclusively bi- or oligo-lamellar ( Figure 1 DF). For up to 6.6×10 13 At least a portion of the GNPs are located inside the formed vesicles, with an Au / L ratio of 100 particles / μmol lipid. In addition, LNPs also advantageously contain large aqueous compartments, which provide the potential for loading small molecule drugs.

[0260] Figure 1 The cryo-transmission images shown in DE show that the GNPs of internalization are located at the obvious connection point between the internal and external vesicles comprising double-layer and oligo-layer LNPs. At least a portion of ionizable cationic lipids can also be compounded with negatively charged GNPs, in the same manner as the ionizable lipids and the siRNA compounded in the LNPs of encapsulated siRNA. Any excessive ionizable lipids not combined with siRNA adopt neutral oil form at pH 7.4, which is separated into hydrophobic domains. Therefore, it is expected that the outer lipid bilayer of hybrid LNP-GNP at pH 7.4 will contain little or no ionizable lipids and therefore do not show net positive surface charge. As shown in Table 1, the ζ-potential of the hybrid LNP-GNP system prepared under multiple Au / L ratios was determined in diluted (×0.25) PBS. As mentioned previously, the negative ζ-potential observed for LNP-GNPs at pH 7.4 is similar to that obtained for the DSPC / Chol system determined under similar conditions in PBS at pH 7.4, consistent with the idea that there are little or no cationic lipids on the LNP-GNP surface.

[0261] Table 1. LNP-GNPs: GNP content, size and surface charge characteristics.

[0262]

[0263] Table 1. Size and ζ-potential values ​​of LNP-GNPs prepared at various Au / L ratios. The LNP lipid composition was DODAP / DSPC / Chol / PEG-DSPE, 10 / 49 / 40 / 1 mol / mol, and GNP encapsulation was performed as shown in the method. The LNPs produced after mixing at pH 4 and dialyzing against PBS at pH 7.4 were measured. ζ-potential measurements were performed in diluted (×0.25) PBS. The standard deviation values ​​of repeated measurements are shown in brackets.

[0264] As mentioned above, cryo-transmission studies of the preparations listed in Table 1 showed that for 13 For the LNP-GNP system prepared with an Au / L ratio of 8.8×10 particles / μmol lipid, there was no evidence of free GNPs, however, for the 13 In the system prepared with 1000 particles / μmol lipid, the aggregated GNPs were obvious ( Figure 1 BD). The Au / L ratio was increased from 2.2×10 13 Increased to 6.6×10 13 particles / μmol lipid did not affect the average size and surface charge properties of LNP-GNPs; however, further increasing to 8.8×10 13 The increase in size and polydispersity and the apparent increase in net negative surface charge resulted in an increase in the number of particles / μmol. In the latter case, the data may reflect the coexistence of LNP-GNPs with (negatively charged) gold particle aggregates.

[0265] Example 2: LNP systems containing high bilayer lipid levels can be produced that contain highly encapsulated gold nanoparticles

[0266] The inventors next evaluated the GNP encapsulation efficiency achieved by the LNP-GNP system at various Au / L ratios. Quantification of GNPs was performed by surface plasmon resonance absorption measurement (Kreibig, U.; Vollmer, M., Theoretical Considerations. In Optical Properties of Metal Clusters, Springer Berlin Heidelberg: Berlin, Heidelberg, 1995; pp. 13-201; the document is incorporated herein by reference), which is a recognized technique for determining GNPs in the presence of lipids dissolved by detergents such as Triton C-100. For this purpose, a range of 0 to 5.5×10 13A calibration curve of 100 particles / mL was then calculated to determine the corresponding LNP-GNP concentrations present in the LNP-GNP samples before and after removal of the external (unembedded) gold.

[0267] Figure 2 (Inset) shows the LNP-GNP system at an Au / L ratio of 1.1×10 13 Up to 8.8×10 13 UV-VIS absorption spectra of the unencapsulated gold (no detergent added) after removal of the unencapsulated gold at an Au / L ratio of 1.1×10 13 to 6.6×10 13 The encapsulation efficiency of the prepared system was close to 100% in the range of 10 particles / μmol lipid, and the Au / L ratio was increased to 8.8×10 13 particles / μmol lipid will result in ~80% gold internalization ( Figure 3 ), which is consistent with the results of cryo-transmission studies ( Figure 1 BD) are well consistent.

[0268] Example 3: Therapeutic agents can be actively loaded into the aqueous compartment of the hybrid LNP-GNP system.

[0269] This example examines the ability to load drugs into LNPs in addition to GNPs. The most robust method for encapsulating drugs into LNP liposome systems is to establish a pH gradient (the interior is acidic) and then load weak base drugs in response to the pH gradient. More than 50% of the common drugs detailed in the Merck Index are weak bases, making pH loading a generally applicable procedure. An effective method for generating a pH gradient is to embed ammonium sulfate (AS) into the vesicles during formation and then remove the external AS. Ammonium (NH4 + ) can dissociate into NH3 + H + , NH3 can then easily permeate out, leaving H + , and thus establish a pH gradient.

[0270] Table 2. LNPs after drug loading

[0271]

[0272] Table 2. The size of LNP-GNPs determined before and after loading with doxorubicin (D / L 0.1 wt / wt). Measurements were made after removal of ethanol and unembedded AS (pre-loaded samples) and after removal of unembedded drug (loaded samples) as described in the methods. Standard deviation values ​​are shown in brackets.

[0273] There are several possible methods to introduce AS into LNPs during formation. One method involves incorporating high concentrations of AS into the aqueous medium containing GNPs during the mixing stage with lipids in ethanol. However, aqueous dispersions of colloidal gold are sensitive to ionic strength. It was found that the addition of AS solution to GNP dispersions resulted in precipitation. An alternative method used by the present inventors is to add AS after LNP formation at pH 4, since the LNP morphology changes when the pH is increased from pH 4 to pH 7.4 (see Figure 1 A and B) may aid encapsulation, and the initial presence of 25% ethanol (v / v) may increase the permeability of the membrane sufficiently to allow AS to penetrate the preformed LNPs. Therefore, the inventors added concentrated (up to 1.8 M) AS aliquots to lipid dispersions containing GNPs at pH 4, followed by dialysis against PBS (see Methods for details).

[0274] The loading study was conducted in two phases. The first phase was to determine how much AS could be encapsulated using the post-formulation AS addition protocol, where concentrated AS aliquots were added dropwise at pH 4 to a sample containing GNPs (Au / L ratio 2.2 × 10 13 The AS was then added to the hybrid LNPs (particles / μmol) to achieve final AS concentrations of 300 mM, 450 mM and 600 mM in solution. The dispersion was dialyzed against PBS to remove residual ethanol, increase the pH and remove unentrapped AS. The resulting LNP GNP system was then dissolved in the presence of a detergent and the ammonium and lipid content was determined.

[0275] like Figure 4 As shown in Figure A, at pH 4, ammonium entrapment levels of 0.24 to 0.28 mol / mol lipid were achieved for initial AS concentrations ranging from 300 mM to 450 mM. In terms of entrapment efficiency, these ammonium entrapment levels reflect a steady decrease in entrapment efficiency with increasing initial AS concentration ( Figure 4 B). Importantly, the presence of AS during the dialysis step did not substantially affect the size or polydispersity of the LNPs, with only a slight increase in size being observed (Table 2).

[0276] Next, it was confirmed that the embedded AS was sufficient to drive the loading of the active loadable therapeutic agent (in this case, a weak base drug). The representative weak base drug selected was the anticancer drug doxorubicin, because doxorubicin can be loaded into the liposome LNP system at a high level, so that the drug is precipitated inside the LNP to form nanocrystals that can be easily imaged by cryo-transmission. Hybrid LNP-GNP samples (pH 7.4) containing AS were prepared as described above, and aliquots of doxorubicin solution were added, and the formulations were cultivated at 60°C using the established doxorubicin loading scheme. The time course of doxorubicin absorption in the hybrid LNP GNP AS system prepared in the presence of 300, 450 and 600mM AS was determined using an initial drug-lipid ratio of 0.1 (wt / wt), and the time course of doxorubicin absorption in the hybrid LNP GNP AS system prepared in the presence of 300, 450 and 600mM AS was determined using the ... Figure 5 For all samples, nearly 100% doxorubicin encapsulation efficiency was achieved within a few minutes of incubation ( Figure 5 A).

[0277] Each doxorubicin molecule accumulated consumes a proton when it reaches the acidic interior, thereby reducing the internal buffering capacity of AS. If the drug-lipid ratio in the initial incubation medium is too high, the buffering capacity will be exhausted and the encapsulation efficiency will be reduced. Therefore, we studied 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. Figure 5 As shown in Figure 2, varying the drug-lipid ratio between 0.05 and 0.1 resulted in effective 100% encapsulation within 5 minutes of incubation. However, if the initial drug-lipid ratio was increased to 0.2, the encapsulation efficiency was significantly reduced. Drug loading resulted in an increase in light particle size as measured by DLS (Table 2).

[0278] like Figure 5 As shown, the doxorubicin loading properties of the hybrid LNP-GNP-AS system are similar to those of reported liposomal doxorubicin formulations (such as Doxil TM ) had comparable loading properties, but had the further advantage of encapsulating GNPs. At high levels of encapsulation, doxorubicin precipitated into fiber bundle nanocrystals, which could be detected on cryo-transmission micrographs. Thus, LNP-GNPs (Au / L 2.2×10 2.0 ... 13 Cryo-transmission studies of drug-loaded LNP-GNPs were performed in a 1:1 (0.1 particles / μmol) formulation to detect these nanocrystals.

[0279] like Figure 6As shown, structures reflecting the precipitated drug can be observed in the hybrid LNPs (indicated by arrows), which give rise to the observed elongated morphology of the LNPs (compared to the more spherical LNP structures observed in the absence of drug, see Figure 1 B). This deformation may explain the slight size increase of the drug-loaded LNP-GNPs shown in DLS. In summary, the particles showing unique drug precipitation formation represent the characteristic "coffee bean" appearance observed earlier for a variety of liposomal doxorubicin formulations. Advantageously, the data show the presence of both gold and therapeutic agent in the same lipid nanoparticle.

[0280] Example 4: Hydrophobic prodrugs can be incorporated into hybrid LNP-GNP systems

[0281] The present embodiment has been tested the ability of hydrophobic drugs loaded into the LNP containing GNP.The lipid characteristics of lipophilic prodrugs can be easily loaded into the LNP system by simply mixing with lipid components.For example, dexamethasone prodrug INT-D034 is incorporated into the LNP-GNP system.The preparation of LNP-GNP system is as previously described and modified.INT-D034, ionizable or cationic lipids, DSPC, cholesterol and PEG-DSPE are mixed in ethanol with a mol ratio of 10 / 10 / 43 / 36 / 1.Use centrifugal concentrator to concentrate aqueous gold colloid (nominal particle diameter is 5nm, and particle concentration is 5.5mg / ml, as provided by the manufacturer), then the concentrated dispersion of appropriate volume is dissolved in 25mM sodium acetate buffer (pH 4.0) to realize desired GNP number density level. Lipids dissolved in ethanol and GNP aqueous dispersions were pumped by two syringe pumps (volume flow ratio of 3:1 (water to ethanol) with pump rates of 15 and 5 ml / min, respectively) and mixed in a "T"-type junction, where two syringes containing organic and aqueous streams were connected to the junction connector. The preparation was dialyzed against PBS to remove residual ethanol. Subsequently, the physicochemical properties of the LNPs prepared as described above were characterized. After buffer exchange to phosphate-buffered saline, the particle size was determined by dynamic light scattering using a Malvern Zetasizer Nano ZS (Malvern, UK). Lipid concentration was determined by measuring total cholesterol using a cholesterol E enzymatic assay kit from WakoChemicals USA (Richmond, VA). INT-D034 encapsulation was determined using UPLC.

[0282] Table 3 below shows that high encapsulation efficiency of GNPs and prodrugs was observed in LNPs.

[0283] Table 3. LNPs containing 5 nm GNPs and INT-D034*

[0284]

[0285]

[0286] (**)3-(Diethylamino)propyl (±)-syn-9,10-dilinoleyloxystearate [INT-A002]

[0287] (***)3-[(±)-syn-9,10-(bis-linoleyloxy)octadecanoyloxy]-N,N-diethyl-N-methylpropane-1-ammonium iodide [INT-A01 7]

[0288] Example 5: Release of an agent of interest (e.g., a drug) using an external stimulus

[0289] After in vitro electromagnetic irradiation, the reagent of interest (e.g., drug) is effectively released from the hybrid LNP containing GNP and the reagent of interest - a hybrid LNP formulation containing GNP and doxorubicin (as an exemplary reagent of interest) was prepared as described above. The physical properties of LNP, such as size / polydispersity and reagent encapsulation efficiency, were determined by dynamic light scattering and UPLC, respectively. The morphology of LNP was analyzed by cryo-transmission.

[0290] A variety of energy sources have been used to trigger drug delivery and release. GNP systems can produce triggered release because they can "explode" in response to high-energy pulsed laser irradiation (e.g., see: 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 destroying the LNP film or structure and promoting drug release. The wavelength of the desired light source depends on the size of the GNP (usually in the range of 200nm to 1000nm).

[0291] Breast cancer cell lines MCF-7 and MDA-MB-231 were incubated with different concentrations of LNPs (10, 50, 100 μg / ml lipid) and placed under light. TM AQ ueous One Solution Cell Proliferation Assay (Promega) or resazurin-based PrestoBlue TMCell viability was measured using the RAPID® assay (ThermoFisher). A decrease in cell viability compared to non-irradiated control cells indicated triggered release of doxorubicin.

[0292] After electromagnetic irradiation in vivo, the reagent of interest (e.g., drug) is effectively released from the hybrid LNP containing GNP and the reagent of interest - a hybrid LNP formulation containing GNP and doxorubicin (as an exemplary reagent of interest) was prepared as described above. The physical properties of LNP, such as size / polydispersity and drug encapsulation efficiency, were determined by dynamic light scattering and UPLC, respectively. The morphology of LNP was analyzed by cryo-transmission.

[0293] The effect of triggered doxorubicin release on the antitumor efficacy of the LNP system was evaluated in a murine xenograft model. MCF-7 or MDA-MB-231 cells were injected subcutaneously into the flank of Balb / c nude mice. Once the tumors reached a standard size (≈100 mm 3 ), 8 mice in each treatment group were intravenously injected with 3 increasing doses of hybrid LNP formulations. Electromagnetic irradiation using high-energy pulsed lasers was applied to the tumor 12 to 24 hours after LNP injection. The wavelength of the required light source depends on the size of the GNP (usually in the range of 200nm to 1000nm).

[0294] Antitumor efficacy was assessed by measuring tumor size with a caliper and data were plotted as median tumor volume (tumor volume (mm 3 ) = length x width 2) / 2). Inhibition or reduction of tumor size compared to non-irradiated control animals indicates successful in vivo triggering of doxorubicin release. Repeated injections of LNPs followed by electromagnetic irradiation were evaluated to compare anti-tumor effects.

[0295] Example 6. Design and synthesis of LNPs loaded with pH gradient loading agents (Dox) and gold nanoparticles (AuNPs)

[0296] In order to produce LNPs that can encapsulate hydrophobic negatively charged gold nanoparticles and hydrophilic drugs (such as Dox), the inventors used a LNP formulation of DODAP / DSPC / Chol / PEG-DSPE, 10 / 49 / 40 / 1 (mol / mol). The molar amounts of ionizable lipids (DODAP) and auxiliary phospholipids (DSPC) were selected so that LNPs adopt a double-layer organization.

[0297] Dox is encapsulated in LNPs with a transmembrane pH gradient with an acidic interior. To this end, the inventors have developed a scheme in which ammonium sulfate (AS) is encapsulated in an aqueous core after AuNPs are incorporated into lipid nanoparticles, thereby creating a pH gradient between the interior and exterior of the lipid nanoparticles. The process of forming lipid nanoparticles follows several steps. In the initial step, lipids are dissolved in ethanol. Subsequently, the resulting solution is mixed into an aqueous medium at pH 4, which contains negatively charged AuNPs at a concentration of 3:1 aqueous medium:ethanol. This is the encapsulation step of AuNPs. Subsequently, a high concentration of 450mM ammonium sulfate aliquots are added. Finally, the mixture is dialyzed against saline to increase pH and remove ethanol. Then, these resulting lipid nanoparticles show an outer layer consisting almost entirely of Chol, DSPC, and PEG.

[0298] exist Figure 6 The obtained LNPs were observed by cryo-transmission imaging in A and 6B. They showed spherical shapes with a diameter close to 100 nm. Figure 6 As schematically shown in C, Dox is present in the aqueous core of the LNP, while AuNP clusters are present in the outer layer. Figure 6 The physicochemical properties of different LNPs (with or without GNPs and Dox) prepared are provided in the table shown in D. The diameters of the LNPs are very close in different types of samples. However, when components such as Dox or GNPs are encapsulated, a slight increase is observed. The negative potential obtained is mainly related to the PEG layer on the outer surface of the LNP. The PEG layer ensures the colloidal stability of the lipid nanoparticles in solution. Comparing different samples, the values ​​are different due to the presence of Dox with a positive potential and gold nanoparticles with a negative potential due to their surface coating.

[0299] The fluorescence of Dox is a property used for observation, and Figure 6 Part of the emission spectrum, using filters for excitation at the appropriate wavelength (505-555 nm), is shown in E. Even when encapsulated in LNPs containing non-fluorescent AuNPs, it is possible to observe fluorescence of Dox. Figure 6 The UV-VIS absorption spectrum of LNPs in F shows a Dox absorption peak around 500 nm and a gold nanoparticle-related peak around 540 nm, especially visible on the LNP / AuNP sample. This suggests that it is possible to obtain a resonance effect on LNPs by irradiating with laser light around 530 nm.

[0300] Example 7: Release of active loaded agent (Dox) from loaded LNP / AuNP / Dox after nanosecond laser irradiation

[0301] This experiment demonstrated that laser irradiation can release Dox from LNPs in the MDA231 breast cancer cell line. Several irradiation protocols were performed on the cells by incubating with different LNP samples and controls. When these LNPs encounter cells, they enter the cells via endocytosis. Fig. 7A It was observed that on the non-irradiated samples with LNP / AuNP / Dox, there were fluorescent red spots inside the cells, indicating the aggregation of LNPs in the endosomes. Fig. 7A As shown, when the LNP contained AuNP and Dox, on the image of the irradiated sample, when Dox was released from the LNP, it penetrated the core and caused it to fluoresce strongly.

[0302] Fig. 7A -C shows representative fluorescence images of Dox release from LNP / AuNP / Dox triggered using nanosecond laser and compared with control experiments with LNP / Dox with and without irradiation and LNP / AuNp / Dox without irradiation. In each of these protocols, a washing step was implemented to remove the LNPs present in the solution and thus reduce the fluorescence background. Experiments were performed with different incubation times of 4 hours and 15 minutes before irradiation and washing steps, as well as irradiation before and after the washing steps. For controls without irradiation, LNP / Dox or LNP / AuNP / Dox diluted in culture medium to a concentration of 50 μg / mL (in LNP) or 5 μg / mL (in Dox) were incubated for 4 hours or 15 minutes according to the protocol. By applying Figure 7B The scheme shown in is used to study the influence of the time of irradiation (in the presence or absence of LNP in the surrounding medium). The sample is irradiated after a minimum incubation time of 15 minutes (the time when LNP is diffused and begins to penetrate the cell). The time of irradiation does not affect the fluorescence intensity of the cell, and therefore does not affect the amount of Dox released to reach the nucleus. In fact, as long as all irradiations are carried out within the same hour, it can be verified that all results are comparable. Only the incubation time, i.e. the time between adding LNP and washing the cells, seems to be an important factor affecting the amount of Dox released. Fluorescence images are taken after a waiting time of 20 hours, so that the Dox released by the laser triggering can diffuse from the LNP to the nucleus.

[0303] Fig. 7AIt is shown that laser-triggered Dox release occurs only in the presence of both AuNPs and irradiation. The images of the LNP / Dox control samples are similar whether or not irradiation is performed, and the laser does not release the drug, which remains encapsulated in the LNP. Drug delivery results can only be obtained when irradiation is combined with the presence of gold nanoparticles in the lipid nanoparticles. When both are present, it is possible to observe that the nucleus is highly fluorescent, which is a sign that Dox is released from the lipid nanoparticles and reaches the nucleus. Therefore, during irradiation, the gold nanoparticles absorb the laser energy, resulting in the release of the cargo contained in the LNP.

[0304] Figure 7B The second scheme shows that the time of irradiation (before or after washing) does not affect the amount of Dox delivered to the nucleus. Irradiation only affects LNPs that are already inside the cell (entered by endocytosis), not LNPs that are still in solution.

[0305] also, Figure 7C It is shown that even a short 15-minute period of LNP incubation with cells is sufficient for LNP to enter cells by endocytosis. Only when there is irradiation and gold nanoparticles in the outer layer of lipid nanoparticles, the strong fluorescence of the nucleus is visible, which indicates that Dox has been released. Note that for all these samples, all cells are still alive, because it is possible to observe the fluorescence of calcein AM, which is a marker of cell viability. However, cells in which Dox can reach the nucleus after irradiation are in the process of cell death.

[0306] Other experiments were performed to quantitatively deliver drugs to the nucleus to evaluate the efficiency of laser irradiation. For this reason, it is believed that the measured nuclear fluorescence intensity is proportional to the release of Dox. The release of laser-triggered Dox from LNP / AuNP / Dox was measured, and the Dox-loaded LNP and Dox-loaded LNP / AuNP in a medium with the same comparable concentration (Dox concentration was 5 μg / mL) were measured. Figure 8Shown are the normalized fluorescence intensities of the unirradiated conditions of LNPs containing Dox for a variety of irradiation and scanning parameters. Note that even under irradiation, when GNPs are not present in the LNPs, these normalized intensities remain relatively constant, confirming that GNPs play an essential role in Dox release. Two laser scanning conditions represented by speeds 1 and 2 were used, in which each LNP / AuNP / Dox had no (speed 1) and a certain (speed 2) overlap between consecutive pulses. At speed 1, when compared to the LNP / Dox irradiated at this fluence, it was found that the irradiation energy threshold of 14.2 μJ effectively induced 6 times the release of Dox from LNP / AuNP / Dox. For the optimization of laser scanning parameters, speed 2 (50 μm / s and a step size of 5 μm between scan lines) was used to enable a small number of laser pulses to overlap each other in space, thereby maximizing Dox release. When the speed was increased to 100 μm / s and 10 μm step size (Speed ​​2), the pulses did not overlap in space, resulting in a release reduction of about 30%. Under optimal conditions, irradiation of LNP / AuNP / Dox resulted in an 11-fold increase in release compared to the non-irradiated LNP / Dox sample.

[0307] Example 8: Release of Actively Loaded Reagents (Dox) from LNP / AuNP / Dox after Femtosecond Laser Irradiation The present inventors also explored the possibility of inducing Dox release from LNP / AuNP / Dox using a femtosecond laser (Spitfire, 800nm, 55fs, 1kHz repetition rate, 35μm spot diameter). When compared to a nanosecond laser at 532nm, a femtosecond laser irradiates at a wavelength away from the Dox absorption peak, thereby minimizing the undesirable photochemical reaction of Dox. In addition, irradiation in the weak absorption biological window (Barbora et al., 2021, PLoS One, Vol. 16, No. 1, pp. e0245350-e0245350; Dabrowski et al., 2016, Coordination Chemistry Reviews, Vol. 325, pp. 67-101; Algorri et al., 2021, Cancers, Vol. 13, No. 14, p. 3484) will lead to a much greater penetration depth for in vivo applications. Despite the non-resonance of 800 nm irradiation with the plasmon peak of 5 nm AuNPs, plasmon-mediated nanobubble formation may occur, as has been shown by Boulais et al. (see Nano Lett, Vol. 12, No. 9, pp. 4763-9, 2012), leading to perforation of the LNP lipid bilayer and release of Dox.

[0308] Fig.9A (bottom) and Fig. 9CThe irradiation protocol provided in is similar to that used with nanosecond lasers, and the results show that this laser can indeed also trigger the release of Dox from LNP / AuNP / Dox. 2 At a fluence of 1.5 and by spatially overlapping laser pulses (i.e., a speed of 3 cm / s and a step size of 30 μm cm between each scan line), Fig.9A Strong red fluorescence was observed. Fig. 9B Shown are the fluorescence intensities normalized to the non-irradiated conditions on LNPs containing Dox for various irradiation and scanning parameters. Fig. 9B As shown in Figure 2, when each pulse overlaps with each other spatially, the threshold for inducing Dox release is 61 mJ / cm 2 , and at 73mJ / cm 2 Under those conditions, each nanoparticle received at most one pulse. Under the best conditions, at the lowest speed, at 73 mJ / cm 2 At a fluence of 1.5 Å, a 3-fold increase was observed between irradiated LNP / Dox and LNP / AuNP / Dox. Using this laser, under optimal conditions, a 7.5-fold increase was obtained between non-irradiated LNP / Dox and irradiated LNP / AuNP / Dox.

[0309] The above description is intended to illustrate the embodiments of the present invention, but is by no means intended to limit the scope of the present invention.

[0310] 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 shall prevail.

Claims

1. A lipid nanoparticle comprising: Helper lipids and ionizable lipids, wherein The ionizable lipid is present in an amount of 2 mol% to 30 mol% relative to the total lipids; at least one lipid layer surrounding an interior having at least one aqueous portion; Encapsulated inorganic particles; and The agent of interest, wherein the agent of interest is a hydrophilic agent present in the at least one aqueous portion or a lipophilic agent present in the at least one lipid layer.

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

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

4. The lipid nanoparticle according to claim 2 or 3, wherein: The at least one aqueous portion is acidic and the reagent of interest is a weak base; or The at least one aqueous portion is basic and the reagent of interest is a weak acid; Optionally, the reagent of interest is pH gradient loadable.

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

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

7. The lipid nanoparticle according to 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, wherein The lipid nanoparticles have an average diameter of 50 to 200 nm, optionally an average diameter of 70 to 150 nm.

9. The lipid nanoparticle according to any one of claims 1 to 8, wherein The at least one lipid layer is bi- or multi-layered.

10. The lipid nanoparticle according to claim 9, wherein The nanoparticle further comprises at least one of a sterol and a hydrophilic polymer-lipid conjugate, optionally 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 according to any one of claims 1 to 11, wherein The helper lipid is present at a concentration of at least 20 mol%, alternatively at least 30 mol%, alternatively at least 35 mol%, alternatively at least 40 mol% or alternatively at least 45 mol%.

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

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

15. The lipid nanoparticle according to any one of claims 1 to 14, wherein The encapsulated inorganic particles are colloids.

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

17. The lipid nanoparticle according to any one of claims 1 to 16, wherein The inorganic particles are negatively charged, optionally wherein the inorganic particles comprise a negatively charged cap.

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

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

20. The lipid nanoparticle according to any one of claims 1 to 18, wherein: The at least one lipid layer comprises a bilayer surrounding an interior, and the inorganic particle is in the bilayer surrounding the interior; and / or The lipid nanoparticle further comprises an inner lipid core, and the inorganic particle is in the inner lipid core.

21. The lipid nanoparticle according to any one of claims 1 to 20, wherein The encapsulated inorganic particles are metal nanoparticles.

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

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

24. A method for producing lipid nanoparticles encapsulating inorganic particles and an agent of interest, the method comprising: (i) combining two separate streams of 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 encapsulating the inorganic particles in the combined stream; (ii) introducing a loading medium into the external solution of the lipid nanoparticle thus formed, the external solution comprising the solvent, and causing the loading medium to be embedded in the lipid nanoparticle, thereby producing a lipid nanoparticle comprising the loading medium and the inorganic particle embedded in its internal compartment; as well as (iii) introducing the agent of interest into an external solution of the lipid nanoparticles including the embedded loading buffer, and allowing the agent of interest to be actively loaded into the lipid nanoparticles in response to the embedded loading medium, thereby producing lipid nanoparticles that embed inorganic particles and the agent of interest.

25. The method according to claim 24, wherein: The pH of the aqueous solution of the inorganic particles in step (i) is less than 5.

5.

26. The method according to claim 24 or 25, wherein: The lipid dissolved in the first formulation comprises an ionizable lipid, optionally a cationic lipid.

27. The method according to claim 26, wherein: The cationic lipid is an amino lipid, and the pH of the aqueous medium is less than the pKa of the cationic lipid, thereby making the cationic lipid charged.

28. The method according to any one of claims 24 to 27, wherein: The first formulation and the second formulation were pumped and mixed in a "T" type junction mixer.

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

30. The method of claim 29, wherein: The loading vehicle is added to the lipid nanoparticles before exchanging the external solution.

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

32. The method according to any one of claims 24 to 31, wherein: The lipids in the first formulation comprise helper lipids and ionizable lipids, wherein the ionizable lipids are present at 2 mol% to 30 mol% relative to the total lipids, alternatively at 5 mol% to 15 mol% relative to the total lipids, and alternatively the helper lipids are present at a concentration of at least 20 mol%, alternatively at least 30 mol%, alternatively at least 35 mol%, alternatively at least 40 mol%, or alternatively at least 45 mol%.

33. A method for producing lipid nanoparticles comprising an inorganic particle core and a lipophilic agent of interest, the method comprising: combining two separate streams of 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 encapsulating the inorganic particle cores in the combined stream; wherein the lipids in the first formulation comprise a helper lipid, an ionizable lipid, and the lipophilic agent of interest; wherein the ionizable lipid is present in an amount of 2 mol % to 30 mol % relative to the total lipids, and optionally in an amount of 5 mol % to 15 mol % relative to the total lipids; and 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 %.

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

36. A method of delivering an agent of interest to a subject using a lipid nanoparticle according to any one of claims 1 to 23, wherein: The lipid nanoparticles are used for administration to the subject, followed by administration of a stimulus to an area of ​​the subject, the stimulus causing the inorganic particles in the lipid nanoparticles to cause the release of the agent of interest from the lipid nanoparticles, optionally the stimulus is electromagnetic radiation, optionally radiation from a light source or by laser.

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

38. The use according to claim 36, wherein The agent of interest is an imaging agent, and wherein the use further comprises imaging a region of the subject.

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

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

41. Use of a lipid nanoparticle according to any one of claims 1 to 23 for treating a mammalian subject in need of treatment, comprising triggered release of an agent at a target site in the body.

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

43. The use according to claim 42, wherein The irradiation has a wavelength that is resonant or non-resonant with the plasmon peak of the metal nanoparticles, and optionally the triggered release is induced by a laser and is resonant.

44. The use according to claim 43, wherein The laser is continuous wave or pulsed. Optionally, the laser is pulsed, and its pulse width is microseconds, nanoseconds, picoseconds or femtoseconds.

45. The use according to claim 44, wherein The radiation is a femtosecond laser having a wavelength that is resonant or non-resonant with the metal nanoparticles, wherein the metal nanoparticles are plasmons.

46. ​​A medical treatment method comprising administering a lipid nanoparticle according to any one of claims 1 to 23 to a mammalian subject in need of such treatment; and subjecting the lipid nanoparticle to irradiation to trigger release of an agent at a target site in the body, optionally the release being caused by irradiation from a light source or by laser.

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