CN113559271A

CN113559271A - Components of macrophage targeting vector system, preparation method and application of macrophage targeting vector system in drug and nucleic acid delivery

Info

Publication number: CN113559271A
Application number: CN202010280531.6A
Authority: CN
Inventors: 章雪晴; 陈起静; 高明珠
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2020-04-10
Filing date: 2020-04-10
Publication date: 2021-10-29

Abstract

The invention provides a carrier system for targeting macrophages. In particular, the invention provides a macrophage-targeting nanoparticle carrier system, including components thereof, methods of preparation, and uses thereof in drug and nucleic acid delivery. The main material of the nano particle provided by the invention has biodegradability, biocompatibility and delayed and sustained release property, and can be injected locally or systemically. In addition, the synthesis method is simple and efficient, and the prepared nano particles contain high-density targeting elements on the surfaces and can deliver drugs and genes to macrophages in a targeted mode with high efficiency. The nano particles rich in the targeting element and the developing agent can be specifically enriched in macrophages and are used for diagnosing and treating diseases.

Description

Components of macrophage targeting vector system, preparation method and application of macrophage targeting vector system in drug and nucleic acid delivery

Technical Field

The invention belongs to the field of medical materials, and particularly relates to a component of a macrophage targeting vector system, a preparation method and application of the macrophage targeting vector system in drug and nucleic acid delivery.

Background

Macrophages are important cells in the innate immune system and play a crucial role in tissue homeostasis and immunity. They can also lead to the development of a variety of pathological processes, including autoimmune, cancer, infectious and inflammatory diseases.

In general, macrophages have two important subgroups: "classical activated macrophages" (M1) and "selectively activated macrophages" (M2), which are present in the inflammatory milieu of various diseases, such as solid tumors, type II diabetes and atherosclerosis. M1s and M2s are believed to be responsible for different functions in these microenvironments. For example, M2s macrophages are notorious for inhibiting tumor immunity and promoting angiogenesis, as compared to M1s, which destroys and phagocytoses tumor cells in the tumor microenvironment. However, in type II diabetes, M2 plays an important role in the development of pancreatic islets, particularly in the proliferation and differentiation of islet beta cells. In contrast, pro-inflammatory factors (e.g., IL-1. beta. and TNF-. alpha.) produced by M1 exacerbate islet beta cell injury. In the context of atherosclerosis, the long-term inflammatory response caused by macrophage-driven deficiencies in inflammation resolution is also a major causative factor. Since macrophage function is broadly associated with the response of a variety of disease states, macrophages are ideal targets for the treatment of a variety of diseases. In this context, it is crucial to develop a highly efficient delivery platform that can target and modulate macrophage function at the site of inflammation, which would be a promising therapeutic strategy for the treatment of a variety of diseases.

Gene transfection is an effective means to analyze and modulate macrophage function at the molecular level. For example, tumor-associated macrophages (TAMs) can suppress tumor immunity and promote angiogenesis. In this case, it has been shown that TAM immune responses and anti-tumor immunity can be activated by insertion of therapeutic genes. However, gene transfection in macrophages is difficult due to degradation of the gene by intracellular reactive oxygen species and lysosomes.

Although viral vectors are popular for their high efficiency of gene transfection, the safety risks associated with viral vectors are a major source of impediments to their widespread use due to off-target immunogenicity, inflammatory responses, and toxicity. To overcome this challenge, non-viral gene transfer methods such as calcium phosphate precipitation, DEAE-dextran, particle bombardment and electroporation have been developed to improve transfection of macrophages. However, these methods tend to have lower gene transfection efficiency or higher cytotoxicity. In addition, non-viral vectors based on cationic lipids and polymers, such as liposomes and poly (. beta. -aminoesters), have also been studied and demonstrated to be effective gene transfection vectors in a variety of cells, but not for macrophages.

Therefore, there is an urgent need in the art to develop a targeting vector system capable of efficiently targeting macrophages, thereby efficiently transfecting macrophages and/or delivering drugs.

Disclosure of Invention

The invention aims to provide a targeting vector system capable of efficiently targeting macrophages, so that efficient transfection and/or drug delivery can be carried out on the macrophages.

In a first aspect of the invention there is provided a nanoparticle for drug and gene delivery targeting macrophages, the nanoparticle comprising:

(i) a polymer segment for forming a matrix of the nanoparticle;

(ii) a targeting element for targeting macrophages, the targeting element being located on the outer surface of the nanoparticle;

wherein the targeting element is selected from the group consisting of:

(Y1) a macrophage-targeting monosaccharide glycoside (preferably, mannose, galactose, or a combination thereof);

(Y2) macrophage targeting peptide fragments (preferably, LyP-1(CRKRLDRNC), CREAK, Collagen IV (KLWVLPKGGGC), or combinations thereof);

(Y3) a glycan (preferably, dextran, mannan, or a combination thereof) targeting macrophage function;

(Y4) any combination of the above Y1 to Y3;

(iii) an optional developing element (i.e., the developing element may or may not be present), the developing element being located on the outer surface or within the nanoparticle; and

(iv) a linking element having one end attached to the polymer segment and the other end attached to the targeting element and/or the visualization element;

with the proviso that,

when the targeting element comprises Y3 and the visualization element is absent, the linking element may be absent, the biodegradable polymer segment is linked to Y3;

when the targeting element comprises Y3 and the visualization element is present, the linking element may be absent, the biodegradable polymer segment and the visualization element are attached to Y3.

In another preferred embodiment, the polymer segment is biodegradable.

In another preferred embodiment, the nanoparticle is targeted to and endocytosed by macrophages by the targeting element.

In another preferred embodiment, the particle size of the nanoparticles is 50-500nm, preferably 70-300 nm.

In another preferred embodiment, the nanoparticle further comprises (v) a loaded drug, wherein the loaded drug is distributed or coated on the matrix of the nanoparticle.

In another preferred embodiment, the nanoparticle further comprises (vi) lipid molecules, and the lipid molecules are distributed or coated on the outer surface or inside of the nanoparticle.

In another preferred embodiment, the nanoparticle comprises a complex formed by a plurality of nanoparticle units.

In another preferred embodiment, the nanoparticle unit comprises: a polymer segment, a targeting element for targeting macrophages, optionally a visualization element, and a linking element for linking the polymer segment and the targeting element together and/or linking the polymer segment and the visualization element together.

In another preferred embodiment, the macrophage targeting element is component Y1, Y2, or a combination thereof.

In another preferred embodiment, the nanoparticle unit comprises: a polymer segment, a targeting element for targeting macrophages, and optionally an imaging element, wherein the targeting element comprises component Y3 and the polymer segment and optional imaging element are attached to the targeting element.

In another preferred embodiment, the macrophage targeting element is a component of dextran, mannan, or a combination thereof.

In another preferred embodiment, the nanoparticle unit has a structure selected from the group consisting of: formula I, formula II or formula III or formula IV;

Z1-Z2-Z3a (formula I)

Z1-Z2-Z3b (formula II)

(Z1) m-Z4- (Z3) n (formula III)

(Z1-Z2-Z3) p-Z4 (formula IV)

Wherein the content of the first and second substances,

z1 is a polymer segment;

z2 is a linking element;

z3a is component Y1, component Y2 or a combination thereof that targets macrophage function;

z3b is a developer;

z3 can be Z3a, Z3b, or a combination of Z3a and Z3b in any ratio;

z4 is macrophage targeting glycan component Y3;

m is an integer of 1 or more (preferably 1 to 432);

n is an integer of 0 or more (preferably 0, or 1 to 432);

p is an integer of 1 or more (preferably 1 to 432)

"-" denotes a chemical bond.

In another preferred embodiment, the nanoparticle unit further comprises a drug, and the drug is distributed or coated on the inner or outer surface of the nanoparticle unit.

In another preferred embodiment, the nanoparticle unit further comprises a lipid molecule, wherein the lipid molecule is compounded with the drug and distributed or coated on the inner or outer surface of the nanoparticle.

In another preferred embodiment, the polymer segment or Z1 is biocompatible and biodegradable.

In another preferred embodiment, the polymer segment or Z1 is selected from the group consisting of: polylactic acid (DL-PLA), polylactic-polyglycolic acid copolymer (PLGA), glycolide-lactide copolymer (PLCG), Polycaprolactone (PCL), polyorthoesters, polyanhydrides, polyphosphazenes, poly-beta-amino esters (PBAE), poly (alpha-hydroxy acids), lactide/glycolide copolymers (PLGA or PLG) including lactide/glycolide copolymers, D-lactide/glycolide copolymers, L-lactide/glycolide copolymers and D, L-lactide/glycolide copolymers, Polyglycolide (PG), polyorthoester(s) (POE), polyethylene glycol (PEG), PEG200, PEG300, PEG400, PEG500, PEG550, PEG600, PEG700, PEG800, PEG900, PEG1000, PEG1450, PEG3350, PEG4500, PEG8000, conjugates of poly (alpha-hydroxy acids), aspirin (polyasirrinpis), polyphosphagens, D-lactide, D, L-lactide-caprolactone, D, L-lactide-glycolide-caprolactone (DL-G-CL), dextran, vinylpyrrolidone, polyvinyl alcohol (PVA), methacrylate, poly (N-isopropylacrylamide), SAIB (sucrose acetate isobutyrate) hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxyethylmethylcellulose, carboxymethylcellulose or a salt thereof, carbopol, poly (hydroxyethyl methacrylate), poly (methoxyethyl methacrylate), poly (methoxyethoxy-ethyl methacrylate), polymethyl methacrylate (PMMA), Methyl Methacrylate (MMA), PVA-G-PLGA, PEGT-PBT copolymer (multi-active), PEO-PPO-PEO (pluronics)), (L-lactide-co-polymer (poly-co-poly-block-co-block-polymer (poly-block-co-block-polymer (poly-block-co-block-polymer (poly-block-polymer (poly-block-co-block-polymer (poly-block-co-polymer (poly-block-co-block-co-polymer-co-polymer (poly-block-co-block-co-polymer (poly-co-block-co-block-co-polymer (poly-co-block-co-block-co-block-polymer (poly-block-co-block-co-poly (poly-co-block-co-block-co-block-co-block-co-block-co-block-co-block-co-block-co-block-co-block-co-block, PEO-PPO-PAA copolymer, PLGA-PEO-PLGA, PEG-PLG, PLA-PLGA, poloxamer 407, PEG-PLGA-PEG triblock copolymer, or their block copolymer with polyethylene glycol (PEG), or one or more of the above polymers or copolymers.

In another preferred embodiment, the polymer segment or Z1 is polylactic-co-glycolic acid (PLGA).

In another preferred embodiment, said attachment element or Z2 is selected from the group consisting of: chemical bonds, PEG, PVA, PPO, or block copolymers thereof, or combinations thereof.

In another preferred embodiment, the chemical bond includes a chemical bond that can be hydrolyzed or enzymatically cleaved (e.g., an ester bond, an amide bond, etc.) or a chemical bond that can be cleaved by light or heat.

In another preferred embodiment, the linking element comprises poly (propylene glycol) or poly (ethylene oxide) (also known as poly (ethylene glycol) PEG).

In another preferred example, the component Y1 having a function of targeting macrophages includes a monosaccharide glycoside structure.

In another preferred embodiment, said component Y2 having a function of targeting macrophages comprises a polypeptide comprising LyP-1(CRKRLDRNC), CREAK, Collagen IV (KLWVLPKGGGC), or a combination thereof.

In another preferred embodiment, the glycan component Y3 with macrophage targeting function comprises dextran, mannan, or a combination thereof.

In another preferred embodiment, the developer or Z3b is selected from the group consisting of: a fluorescent probe, a magnetic resonance imaging contrast agent, a radioactive element, a heavy metal, or a combination thereof.

In another preferred embodiment, the drug (component v) comprises a small molecule or a biological macromolecule.

In another preferred embodiment, the drug is selected from the group consisting of: nucleic acids, ribonucleic acid protein complexes, vaccines, proteins, Insulin (e.g., Insulin glargine, etc.), polypeptides, glucagon-like peptide-1 (GLP-1) and analogs thereof, immunogenic compositions, antigens, exosomes, or combinations thereof.

In another preferred embodiment, the nucleic acid is selected from the group consisting of: pDNA, siRNA, mRNA, miRNA, shRNA, non-coding RNA, or a combination thereof.

In another preferred embodiment, the vaccine is selected from the group consisting of: deactivating viral particles, extinguishing viral particles, pseudovirus-like particles, or a combination thereof.

In another preferred embodiment, the protein is selected from the group consisting of: a tumor necrosis factor inhibitor Etanercept, a fusion protein, a recombinase, and a recombinant protein, or a combination thereof.

In another preferred embodiment, the medicament is a medicament for treating a disease or condition selected from the group consisting of: lenalidomide, inhibitors of viral synthesis and assembly Ledipasvir, statins lipid lowering drugs, curcumin and its analogs, Tofacitinib and its salts, Liver X Receptor antagonists (Liver X Receptor Agonists), narcotic analgesics, anti-inflammatory agents, anti-cancer agents, agents for treating diabetes, agents for treating obesity, or combinations thereof.

In another preferred embodiment, the lipid molecule (component vi) is selected from the group consisting of: phospholipids, cholesterol and its derivatives, fatty acids and their esters, lipid compounds, or combinations thereof.

In another preferred embodiment, the phospholipid is a phospholipid having a structure selected from the group consisting of Phosphatidylethanolamine (PE), Phosphatidylethanolamine (PC), and Phosphatidylserine (PS).

In another preferred embodiment, the lipid compound comprises a lipid compound, a lipidoid, or a combination thereof.

In another preferred embodiment, the lipid molecule is lipid compound G0-C14.

In another preferred embodiment, the structure of formula I is:

(PLGA-PEG-mannose, PPM), or

(PLGA-PEG-galactose, PPG).

In another preferred embodiment, the structure of formula III is:

(PLGA-dextran, PD).

In another preferred embodiment, the structure of formula III is:

(PLGA-Glucan-mannose, PDM).

In a second aspect of the invention, there is provided a pharmaceutical composition comprising:

(i) a nanoparticle according to the first aspect of the present invention, wherein said nanoparticle comprises a drug loaded, said drug loaded being distributed or entrapped on the outer surface or within the interior of said nanoparticle; and

(ii) a pharmaceutically acceptable carrier.

In another preferred embodiment, the drug molecule is embedded in the nanoparticle.

In another preferred embodiment, the composition enters macrophages at a rate of 30% to 90%, preferably 40% to 90%, more preferably 50% to 90%, more preferably 60% to 90%, more preferably 70% to 90%, more preferably 80% to 90%.

In another preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from: solid preparation, liquid preparation or injection.

In another preferred embodiment, the subject to which the pharmaceutical composition is administered is a mammal, preferably a human.

In another preferred embodiment, the dosage form of the pharmaceutical composition is an injection.

In another preferred embodiment, the injection is administered by a method comprising: intravenous injection, intramuscular injection, subcutaneous injection, intracavitary injection, intracrystalline injection, intraocular injection, and the like.

In a third aspect of the present invention, there is provided a method for preparing nanoparticles according to the first aspect of the present invention, comprising the steps of:

(i) providing a nanoparticle unit having a structure selected from the group consisting of: formula I, formula II or formula III or formula IV

Z1-Z2-Z3a (formula I)

Z1-Z2-Z3b (formula II)

(Z1) m-Z4- (Z3) n (formula III)

(Z1-Z2-Z3) p-Z4 (formula IV)

Wherein the content of the first and second substances,

z1 is a polymer segment;

z2 is a linking element;

z3b is a developer;

z3 can be Z3a, Z3b, or a combination of Z3a and Z3b in any ratio;

z4 is macrophage targeting glycan component Y3;

m is an integer of 1 or more (preferably 1 to 432);

n is an integer of 0 or more (preferably 0, or 1 to 432);

p is an integer of 1 or more (preferably 1 to 432)

"-" denotes a chemical bond; and

(ii) mixing the nanoparticle units to obtain nanoparticles according to the first aspect of the invention.

In another preferred embodiment, the nanoparticle unit comprises a drug, and the drug is distributed or coated on the inner or outer surface of the nanoparticle unit.

In another preferred embodiment, the nanoparticle unit further comprises a lipid molecule, and the lipid molecule is compounded with the drug and distributed or coated on the inner or outer surface of the nanoparticle.

In another preferred embodiment, in step (ii), the mixing is carried out in the presence of a stabilizer.

In another preferred embodiment, the stabilizer is selected from the group consisting of: polyvinyl alcohol, polyglycerol fatty acid ester, tween 80, tween 20, Span80, Span60, sodium dodecyl sulfate, or a combination thereof.

In another preferred embodiment, the stabilizer is used in a concentration of 0.1 wt% to 10 wt%.

In another preferred embodiment, the method further comprises step (iii): isolating said nanoparticles.

In another preferred embodiment, in step (iii), the removal of the stabilizer is included.

In another preferred embodiment, the nanoparticles are prepared by a method selected from the group consisting of: a nano-precipitation method, an emulsification-solvent evaporation method, or a combination thereof.

In a fourth aspect of the present invention, there is provided a use of the nanoparticle according to the first aspect of the present invention for the preparation of a medicament for targeting macrophages or for the preparation of a medicament for the treatment of a disease associated with macrophages.

In another preferred embodiment, the macrophage-related disease comprises: diabetes, obesity, cancer, pulmonary fibrosis, cardiovascular disease, inflammatory disease or a combination thereof.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows DMSO-d₆Of (A) PD, (B) PPM and (C) PPG polymers¹H NMR spectrum and (D) GPC curve of PD polymer.

FIG. 2 shows (A) a schematic of macrophage targeting NPs modified with different saccharides; (B) hydrodynamic diameter of NPs prepared by nano-precipitation; (C) TEM images of 1% uranyl acetate stained PPM NPs.

FIG. 3 shows macrophage Raw264.7 with different concentrations of nanoparticles, e.g. 0mg/mL (control, black curve for each histogram), 0.45mg/mL (red curve), 0.90mg/mL (blue curve), 1.60mg/mL (orange curve) and 2.80mg/mL (green curve), flow characterization of cells for NPs uptake after incubation for a certain time, and bar graph characterization of the corresponding endocytosis efficiency (Cy5.5% positive cells). Statistical significance was calculated using the Student's t-test and represents the comparison between targeted and non-targeted NPs at each concentration (. P <0.05,. P <0.01,. P <0.001 and. P < 0.0001). Data are presented as mean ± standard deviation (n ═ 3).

FIG. 4 shows the cytotoxicity of NP against Raw264.7 cells at various concentrations of 0.90mg/mL, 1.60mg/mL, and 2.80 mg/mL. Cell viability is the percentage relative to the control group. Data are presented as mean ± standard deviation (n ═ 5).

FIG. 5 shows (A) a schematic of the self-assembly process of pDNA loaded PPM/G0-C14 hybrid NPs; (B) electrophoretic analysis of mRNA and pDNA mobility in the complex of G0-C14; (C) TEM images of PPNA-loaded PPM/G0-C14 NPs stained with 1% uranyl acetate.

FIG. 6 shows the phagocytic efficiency of macrophages by various concentrations of NP. Data are expressed as percent Cy5.5 positive (Cy5.5+) cells (A) and MFI (fluorescence intensity of Cy5.5 positive cells) (B). The MFI of cy5.5 was calculated using Flowjo software and the values were processed as relative to the control. Data are expressed as mean ± standard deviation (n ═ 3), and statistical differences between targeted NPs and non-targeted PP NPs were determined using Student's t-test. P <0.001, P <0.01, P < 0.05. NS stands for not significant.

FIG. 7 shows the transfection efficiency of Raw264.7 cells mediated by mRNA-loaded hybrid NPs (A and B) and pDNA-loaded hybrid NPs (C and D) at gene concentrations of 0.125, 0.250, 0.500 and 1.000. mu.g/mL, respectively. Data are expressed as percentage of GFP positive cells (a and C) and MFI of GFP (B and D). The MFI of GFP was calculated using Flowjo software and the values were processed as relative to the control. Data are expressed as mean ± standard deviation (n ═ 3) and the degree of statistical difference was calculated by Student's t-test: p <0.001, P <0.01, P < 0.05.

Detailed Description

The present inventors have conducted extensive and intensive studies and extensive screening to develop a series of novel nanoparticles based on PLGA or PLGA-PEG for the first time, the periphery of which is modified with various saccharides (e.g., mannose, galactose, a mixture of mannose and galactose, dextran, etc.). Experimental results show that a series of nano drug delivery systems based on the high molecules and the cationic liposome can enhance phagocytosis of the macrophage to the nano particles, effectively deliver drugs and nucleic acid molecules to the macrophage, and generate obvious drug effect and effectively regulate expression of related genes/proteins. The nano-particle has uniform and stable size, has no cytotoxicity, can efficiently entrap and slowly release drugs and nucleic acid molecules, and can efficiently target macrophages to transfer the drugs and the nucleic acid molecules. The invention provides a series of components of a nano drug delivery system which can target macrophages and deliver drugs and nucleic acid molecules safely and efficiently, a preparation method and application thereof in drug and nucleic acid delivery, provides a drug preparation platform with great potential for prevention and treatment of various diseases, and also provides a safe and effective technical method and tool platform for researching action mechanisms of the macrophages in various inflammation-related diseases. The present invention has been completed based on this finding.

Specifically, the present inventors prepared novel macrophage-targeting nanoparticles (targeting vector system), and studied their cytotoxicity using the CCK-8 assay, and analyzed the phagocytosis efficiency of fluorescently labeled nanoparticles by macrophages by flow cytometry. EGFP mRNA and GFP pDNA were selected as reporter genes, respectively, and gene transfection ability was compared between nanoparticles modified with different sugars and unmodified nanoparticles. Experiments prove that the targeting vector system does not generate any systemic toxicity, can efficiently deliver the medicament and the nucleic acid molecules to the macrophage in a targeting way, effectively increases the phagocytosis of the macrophage to the targeting nanoparticles, the delivered gene expresses more protein, the delivered medicament is slowly released to play the aim of continuous treatment, and various diseases mediated by the macrophage can be effectively treated.

Nanoparticles and method for preparing same

As used herein, the terms "nanoparticle," "nanoparticle complex," "NP," are used interchangeably and refer to the nanoparticles provided herein that target macrophages for drug and gene delivery.

As used herein, the terms "component targeting macrophage function" and "targeting element" are used interchangeably to refer to an element for targeting macrophages that is located on the outer surface of the nanoparticle of the present invention.

In the present invention, there is provided a nanoparticle for drug and gene delivery targeting macrophages, the nanoparticle comprising: (i) a polymer segment for forming a matrix of the nanoparticle; (ii) a targeting element for targeting macrophages, the targeting element being located on the outer surface of the nanoparticle; (iii) an optional developing element (i.e., the developing element may or may not be present), the developing element being located on the outer surface or within the nanoparticle; and (iv) a linking element, one end of which is attached to the polymer segment and the other end of which is attached to the targeting element and/or the visualization element.

In the present invention, the nanoparticle is targeted to and endocytosed by macrophages by the targeting element. In one embodiment, the nanoparticles of the present invention have a particle size of 50 to 500nm, preferably 70 to 300 nm.

Preferably, the targeting element is selected from the group consisting of: (Y1) a macrophage-targeting monosaccharide glycoside (preferably, mannose, galactose, or a combination thereof); (Y2) macrophage targeting peptide (preferably, LyP-1(CRKRLDRNC) (SEQ ID NO:1), CREAK, Collagen IV (KLWVLPKGGGC) (SEQ ID NO:2), or a combination thereof); (Y3) a glycan (preferably, dextran, mannan, or a combination thereof) targeting macrophage function; (Y4) any combination of the above Y1 to Y3.

In the structure of the nanoparticle of the present invention, when the targeting element comprises Y3 and the visualization element is absent, the linking element may be absent, the biodegradable polymer segment is linked to Y3; when the targeting element comprises Y3 and the visualization element is present, the linking element may be absent, the biodegradable polymer segment and the visualization element are attached to Y3.

The nanoparticles provided by the present invention are useful for drug delivery, and when used for drug delivery, the subject materials of the nanoparticles of the present invention are biodegradable.

In one embodiment, the nanoparticle further comprises (v) a loaded drug, said loaded drug being distributed or entrapped within the matrix of said nanoparticle. Preferably, the nanoparticle further comprises (vi) lipid molecules, wherein the lipid molecules are distributed or coated on the outer surface or the inner part of the nanoparticle.

The nanoparticle provided by the invention can be a complex formed by a plurality of nanoparticle units. The nanoparticle unit comprises: a polymer segment, a targeting element for targeting macrophages, optionally a visualization element, and a linking element for linking the polymer segment and the targeting element together and/or linking the polymer segment and the visualization element together.

Specifically, the nanoparticle unit has a structure selected from the group consisting of: formula I, formula II or formula III or formula IV;

Z1-Z2-Z3a (formula I)

Z1-Z2-Z3b (formula II)

(Z1) m-Z4- (Z3) n (formula III)

(Z1-Z2-Z3) p-Z4 (formula IV)

Wherein the content of the first and second substances,

z1 is a polymer segment;

z2 is a linking element;

z3b is a developer;

z3 can be Z3a, Z3b, or a combination of Z3a and Z3b in any ratio;

z4 is macrophage targeting glycan component Y3;

m is an integer of 1 or more (preferably 1 to 432);

n is an integer of 0 or more (preferably 0, or 1 to 432);

p is an integer of 1 or more (preferably 1 to 432)

"-" denotes a chemical bond.

In a preferred embodiment, the structure of formula I is:

(PLGA-PEG-mannose, PPM), or

(PLGA-PEG-galactose, PPG).

In a preferred embodiment, the structure of formula III is:

(PLGA-dextran, PD).

In a preferred embodiment, the structure of formula III is:

(PLGA-Glucan-mannose, PDM).

The compound has good dispersibility and stability in NaCl aqueous solution, PBS aqueous solution or serum, and no precipitation or agglomeration phenomenon.

In the present invention, in formula I, the structural unit consisting of Z1-Z2 may be selected from: oligopeptide nanoparticles, phospholipid nanoliposomes, polysaccharide nanoparticles, polyether nanoparticles, polyester polymer micelles, or combinations thereof. Among them, albumin-based nanoparticles, phospholipid-based nanoparticles, polysaccharide-based nanoparticles, and polyester-based nanoparticles are preferable.

One preferred class of proteinaceous nanoparticles comprises: human serum albumin nanoparticles (HSA), bovine serum albumin nanoparticles (BSA), or a combination thereof.

One preferred class of phospholipid nanoliposomes comprises: phosphatidylcholine (PC) nanoliposomes, Dipalmitoylphosphatidylcholine (DPPC) nanoliposomes, Distearoylphosphatidylcholine (DSPC) nanoliposomes, Dipalmitoylphosphatidylethanolamine (DPPE) nanoliposomes, Distearoylphosphatidylethanolamine (DSPE) nanoliposomes, Dipalmitoylphosphatidylglycerol (DPPG) nanoliposomes, or combinations thereof.

One preferred class of polyester-based nanoparticles comprises: polyethylene glycol-polylactic acid (PEG-PLA) nanoparticles, polyethylene glycol-polylactide glycolide (PEG-PLGA) nanoparticles, polyethylene glycol-polycaprolactone (PEG-PCL) nanoparticles, or a combination thereof.

One preferred class of polysaccharide nanoparticles comprises: chitosan-based nanoparticles.

One preferred class of polyester-based polymer micelles comprises: polyethylene glycol-polylactic acid (PEG-PLA) micelles, polyethylene glycol-polycaprolactone (PEG-PCL) micelles, polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE) micelles, polyethylene glycol-polyethyleneimine (PEG-cl-PEI) micelles, or a combination thereof.

The preparation method of the nano particles mainly comprises the following steps: (1) preparation of the nanoparticle units in the composite and (2) mixing of the nanoparticle units to obtain the nanoparticles of the invention.

The preparation method of the nanoparticle unit can adopt a method well known by a person skilled in the art to prepare the nanoparticle unit.

Preferably, in step (2), the mixing is carried out in the presence of a stabilizer. Wherein the stabilizer is selected from the group consisting of: polyvinyl alcohol, polyglycerol fatty acid ester, tween 80, tween 20, Span80, Span60, sodium dodecyl sulfate, or a combination thereof. Preferably, the stabilizer is used at a concentration of 0.1 wt% to 10 wt%.

In another preferred embodiment, the method further comprises step (iii): isolating the nanoparticles, including removing the stabilizing agent.

In a preferred embodiment, the nanoparticle units can be combined by precipitation to obtain nanoparticles, i.e., the nanoparticles of the present invention.

Medicaments, compositions and methods of administration

The medicament of the invention contains an effective amount of the composition of the invention, and a pharmaceutically acceptable carrier or excipient.

As used herein, the terms "comprising" or "including" include "comprising," consisting essentially of … …, "and" consisting of … …. As used herein, an ingredient of the term "pharmaceutically acceptable" is one that is suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., at a reasonable benefit/risk ratio. As used herein, the term "effective amount" refers to an amount that produces a function or activity in and is acceptable to humans and/or animals.

As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents. The term refers to such pharmaceutical carriers: they are not essential active ingredients per se and are not unduly toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. A thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences, Mack pub.Co., N.J.1991.

The pharmaceutical dosage form of the present invention comprises: solid preparation, liquid preparation or injection. Preferably an injection.

The subject to which the medicament of the invention is administered is a mammal, preferably a human.

In another preferred embodiment of the invention, the medicament or composition of the invention is administered one or more times per day, e.g. 1,2, 3, 4, 5 or 6 times. Wherein routes of administration include, but are not limited to: oral administration, injection administration, intracavity administration, transdermal administration; preferred administration by injection includes: intravenous injection, intramuscular injection, subcutaneous injection, intracavity injection, intracrystalline injection, intraocular injection. The specific dosage for administration of the pharmaceutical or composition of the invention will also take into account factors such as the route of administration, the health of the patient, and the like, which are within the skill of the skilled practitioner. A safe and effective amount of a composition of the invention is generally at least about 10mg or at least 85 mg/kg body weight/day, and in most cases no more than about 200 or no more than about 115 mg/kg body weight/day. The preferred dosage is about 100 mg/kg body weight/day.

The main advantages of the invention include:

1) the main material for constructing the nano particles has biodegradability, biocompatibility and delayed and sustained release property, and can be injected locally or systemically.

2) The synthesis method of the nano-particle is simple and effective, and the prepared nano-particle contains high-density targeting elements on the surface and can deliver drugs and genes to macrophages in a targeted manner with high efficiency.

3) The nano-particles rich in the targeting element and the developing agent can be specifically enriched in macrophages for diagnosis and treatment of diseases.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Experimental Material

Carboxyl terminated PLGA [50:50 Poly (DL-lactide-glycolide) (0.55-0.75dL g)^-1)]Commercially available from loctite Absorbable Polymers (latex Absorbable Polymers).

Heterobifunctional PEG Polymer NH₂-PEG-OH and NH₂PEG-SH (molecular weight 3400Da) was purchased from Lyshan Bio (Laysan Bio, Inc.).

BOC-HN-PEG-NHS and PEG (amino terminated, 3kDa) were purchased from Jenken (Jenkem Technology USA) in the United states.

4-aminophenyl α -D-mannopyranoside is available from synthetic sugars, Inc. (Synthose Inc.).

4-aminophenyl beta-D-pyranosides are available from Shanghai Bibo pharmaceutical technology, Inc.

N-hydroxysuccinimide (NHS), 1-ethyl-3- [ 3-dimethylaminopropyl ] ] carbodiimide (EDC) hydrochloride, N-Diisopropylethylamine (DIPEA) and trifluoroacetic acid (TFA) were purchased from Beijing YinoKa technologies, Inc.

Dextran (MW 70kDa) and polyvinyl alcohol (PVA, MW 20kDa) were purchased from shanghai mclin biochemical technologies, ltd.

4- (dimethylamino) pyridine (DMAP) was purchased from J & K technologies, Shanghai, China.

Anhydrous pyridine, lithium chloride (LiCl), Triethylamine (TEA), anhydrous dimethyl sulfoxide (DMSO), anhydrous Dimethylformamide (DMF), mono-boc protected ethylenediamine (BocEDA) and Cell Counting Kit-8 (Cell Counting Kit-8)/WST-8 were purchased from Morgan Biotech, Inc. of Shanghai.

4-nitrophenyl chloroformate (PNC) methanol was purchased from Shanghai Tatan chemical Co., Ltd.

Sulfocyanine 5.5NHS ester and sulfocyanine 5.5 maleimide were purchased from lumirobe (Lumiprobe).

Cationic ethylene diamine core-Polyamide (PAMAM) dendrimer generation 0 (G0) and 1, 2-epoxytetradecane (C14) were purchased from Sigma-Aldrich.

Fetal Bovine Serum (FBS) was purchased from Biyotime corporation.

Dulbecco's modified Eagle Medium (DMEM; Gibco), fetal bovine serum (FBS; Gibco), streptomycin and penicillin (Thermo-Fisher Scientific) were purchased from Yinyi Weijie (Shanghai) trade company Limited.

Characterization method

Carried out on an Agilent science 500/54Premium Shield instrument¹H NMR analysis the structure of the polymer was characterised and in DMSO-d₆(2.50)、CDCl₃(7.26) or D₂The residual solvent content of O (4.79) is referenced and the chemical shifts are reported. Gel permeation chromatography (GPC, EcoSeC HLC-8320) was used to analyze the molecular weight and dispersion of the polymers. The eluent was DMF containing 0.02M lithium bromide (LiBr) at a flow rate of 1mL/min and a column temperature of 50 ℃. Dynamic Light Scattering (DLS) was performed at a detection angle of 90 ° on Malvern ZS 90. The mean diameter and polydispersity were measured at 25 ℃ and automatically analyzed in cumulative mode using Malvern software. The morphology of the NPs was characterized by transmission electron microscopy (TEM, FEI Tecnai G2 Spirit Bio TWIN). TEM samples were prepared by dropping NP solution (1% w/v) onto a copper grid and stained with 1% uranyl acetate. The fluorescence intensity of RAW264.7 macrophages was analyzed using a flow cytometer (BD lsrfortessa tm) and the data was analyzed using FlowJo software.

Abbreviations

D-70 refers to dextran with a molecular weight of 70kDa (MW 70 kDa);

PNC: 4-nitrophenyl chloroformate;

BocEDA: mono-boc protected ethylenediamine;

experimental methods

Synthesis of PLGA-dextran (PD)

Synthesis of D-70-PNC. D-70(1.0g) and LiCl (2.0g, 2 w/v% DMF) were weighed into a 150mL round bottom flask. Anhydrous DMF (100mL) was added and stirred for 3 min. The suspension was stirred continuously at 90 ℃ for 1h until the suspension became clear. Then cooled to 0 ℃ in an ice-water bath and added sequentially with anhydrous pyridine (1.3mL, 0.015mol) and PNC (3.11g, 0.015 mol). Stirring at 0 deg.C for 4h, precipitating the solution into cold ethanol, washing with diethyl ether for 3 times, vacuum drying for 12h to obtain D-70-PNC with yield of 1.6g (4)0％)。¹H NMR(DMSO-d₆400MHz): 7.54 and 8.28ppm (b, aromatic protons), 5.39 and 5.52ppm (S, dextran glycoside protons with nitrophenyl substituents), 4.9, 4.8 and 4.5ppm (S, dextran hydroxyl protons), 4.7ppm (S, dextran terminal protons), 3.19-3.73 ppm (m, dextran glycoside protons) (FIG. S1).

And (3) synthesizing D-70-Boc. 0.32g LiCl was dissolved in 32mL of anhydrous DMF, 0.4g D-70-PNC was added and dissolved with stirring, nitrogen bubbling was performed, then 3.6g of BocEDA diluted with 2mL of anhydrous DMF was added, nitrogen bubbling was continued for 30min, and stirring was performed for 12h under a nitrogen atmosphere. The reaction solution was precipitated into absolute ethanol, then washed twice with ether and dried in vacuo to give 0.3% product in 80% yield. The nuclear magnetism result of DMSO-d6 as a solvent is as follows:¹H NMR(DMSO-d₆500MHz) 4.50, 4.84 and 4.91ppm (s, dextran hydroxyl protons), 4.66ppm (s, dextran end protons), 3.10-3.89ppm (m, dextran glycoside protons), 3.33-3.52ppm (m, -CH2-CH2-), 1.37ppm (s, -C (CH3) 3).

D-70-NH₂And (4) synthesizing. 0.3g D-70-Boc was dissolved in 3mL deionized water and trifluoroacetic acid (TFA) was added, wherein the TFA concentration was 9% by volume. The mixture was stirred overnight with nitrogen bubbling. The pH was adjusted to neutral with 4M NaOH, then dialyzed against water and freeze-dried to give 0.06g of a white solid in 20% yield. The nuclear magnetism result measured by using heavy water as a solvent is as follows:¹H NMR(D₂o, 500MHz) 4.81ppm (s, dextran terminal protons), 3.31-3.89ppm (m, dextran glycoside protons), 2.97 and 3.46ppm (m, -CH)₂-CH₂-)。

Synthesis of PLGA-dextran (PD). 1mg of PLGA-COOH was weighed into a 25mL reaction flask and dissolved in 3mL of anhydrous DCM. 0.0247g of EDC and 0.014g of NHS were added into the reaction flask, wherein the molar ratio of PLGA-COOH: EDC: NHS was 1:5:5, and the mixture was stirred at room temperature for 2 h. The reaction solution was added dropwise to a mixed solvent of ether/methanol (50/50v/v), centrifuged and redissolved with DCM, added dropwise to a mixed solvent of ether/methanol again, and finally rotary evaporated and dried in vacuo to give the PLGA-NHS intermediate. Dissolving PLGA-NHS with anhydrous DCM, and adding D-70-NH₂Adding N, N-Diisopropylethylamine (DIEA) after the mixture is completely dissolved, wherein PLGA-NHS: D-70-NH₂The molar ratio of DIEA is 1/1.5/10. The solution was stirred at room temperature for 24h to complete the reaction. And precipitating the reaction solution into an ether/methanol mixed solvent, repeating precipitation twice, finally dissolving with DCM, performing rotary evaporation, and drying in vacuum to obtain the product. With CDCl₃The nuclear magnetism result of solvent measurement is as follows:¹H NMR(CDCl₃，500MHz):(5.21ppm(m，-OCH(CH₃)CONH-)，4.91(m，-OCH₂COO-), 3.20-3.74 ppm (m, dextran glycoside protons), 4.67ppm (s, dextran hydroxyl protons), 4.49ppm (s, dextran terminal protons), 1.48(d, -OCH (CH)₃)CONH-)ppm。

Preparation of PLGA-Glucan-Cy5.5 (PD-Cy5.5)

Sulfo-Cyanine 5.5NHS ester (5mg) and PD (50 mg). Dissolved in 4mL of anhydrous DMSO, and then DIPEA (8 μ L) was added. The reaction was stirred at room temperature for 48 hours. PD-Cy5.5 was obtained by dialysis with water (MWCO 10KDa) followed by lyophilization.

Synthesis of PLGA-PEG-mannose (PPM)

And (3) synthesizing tBOC-NH-PEG-mannose. tBOC-NH-PEG-NHS (0.2g, 0.06mmol) and MAN (0.0195g, 0.072mmol) were dissolved in 1mL anhydrous DMF and DIPEA (103. mu.L, 0.6mmol) was added and stirred at room temperature for 48 h. The reaction solution was precipitated into anhydrous diethyl ether, washed with diethyl ether 2 times, and dried in vacuo to give 0.205g of tBOC-NH-PEG-Mannose. The yield was 95%.¹H NMR (DMSO, 400MHz) 7.5, 7.0ppm (d, phenyl protons), 5.26-4.4ppm (m, mannose protons, 3.5ppm (s, -CH2CH2O-), 1.37ppm (s, -C (CH3) 3).

NH₂-synthesis of PEG-mannose. tBOC-NH-PEG-mannose (0.2g) was dissolved in 4mL of DCM containing 50 vol% TFA and stirred at room temperature for 50 min. The reaction solvent was removed by rotary evaporation and the product was dialyzed against water (MW ═ 1KDa) for three days. Finally, NH was obtained by lyophilization₂-PEG-mannose. Yield: 0.15g (75%).¹H NMR (DMSO, 400MHz):7.5, 7.0ppm (d, phenyl proton), 5.26-4.4ppm (m, mannose proton), 3.5ppm (s, -CH)₂CH₂O-)。

Synthesis of PLGA-PEG-mannose (PPM). PLGA (0.2g) was dissolved in dry DCM (1.5 mL). EDC. HCl in 0.5mL of anhydrous DCM (10mg, 0.05mmol) and NHS in 10. mu.L of DMSO (5.7mg, 0.05mmol) were added sequentially. The reaction was stirred at room temperature for 2 hours. PLGA-NHS was obtained by precipitation in cold methanol/ether (50/50v/v), further washed twice and dried by rotary evaporation. PLGA-NHS was dissolved in anhydrous DCM (2mL) and NH was then added₂-PEG-mannose (0.026g) dissolved in the solution. DIPEA (10. mu.L) was added, and the mixture was stirred at room temperature for 48 hours. PLGA-PEG-mannose (PPM) was obtained by precipitation in methanol/diethyl ether (50/50v/v) and washed three times with methanol/diethyl ether and finally dried in vacuo to remove the solvent. Yield: 0.19g (87%).¹H NMR(D₂O, 400MHz):7.5, 7.0ppm (d, phenyl proton), 5.20ppm (m, -OCH (CH)₃)CONH-)，4.91ppm(m，-OCH₂COO-)，3.51ppm(s，-CH₂CH₂O-)，1.48(d，-OCH(CH₃)CONH-)ppm。

Synthesis of PLGA-PEG-galactose (PPG)

And (3) synthesizing tBOC-NH-PEG-galactose. tBOC-NH-PEG-NHS (0.2g, 0.06mmol) and GAL (0.0195g, 0.072mmol) were dissolved in dry DMF (1mL) and DIPEA (103. mu.L, 0.6mmol) was added. The solution was stirred at room temperature for 48 hours. tBOC-NH-PEG-galactose was obtained by precipitation in diethyl ether, further washed twice with diethyl ether and dried in vacuo. Yield: 0.205g (95%).¹H NMR (DMSO, 400MHz): 7.48, 6.95ppm (d, phenyl proton), 5.34-4.1ppm (m, galactose proton), 3.49ppm (s, -CH)₂CH₂O-)，1.37ppm(s，-C(CH₃)₃)。

Synthesis of NH 2-PEG-galactose. tBOC-NH-PEG-galactose (0.2g) was dissolved in 4mL of DCM containing 50 vol% TFA and stirred at room temperature for 50 min. The reaction solvent was removed by rotary evaporation and the product was dialyzed against water (MW ═ 1KDa) for three days. Finally, NH was obtained by lyophilization₂-PEG-galactose. Yield: 0.15g (75%).¹H NMR (DMSO, 400MHz): 7.48, 6.95ppm (d, phenyl protons), 5.34-4.1ppm (m, mannose protons), 3.5ppm (s, -CH)₂CH₂O-)。

Synthesis of PLGA-PEG-galactose (PPG). PLGA (0.2g) was dissolved in dry DCM (1.5 mL). EDC. HCl in 0.5mL of anhydrous DCM (10mg, 0.05mmol) and NHS in 10. mu.L of DMSO (5.7mg, 0.05mmol) were added sequentially. The solution was stirred at room temperature for 2 hours. PLGA-NHS was obtained by precipitation in cold methanol/ether (50/50v/v), further washed twice and dried by rotary evaporation. PLGA-NHS was dissolved in dry DCM (2mL) and NH was then added₂-PEG-galactose (0.026g) was dissolved in the solution. DIPEA (10. mu.L) was added, and the mixture was stirred at room temperature for 48 hours. PLGA-PEG-galactose (PPG) was obtained by precipitation in methanol/diethyl ether (50/50v/v) and washed three times with methanol/diethyl ether and finally dried in vacuo to remove the solvent. Yield: 0.19g (87%).¹H NMR(D₂O, 400MHz): 7.51, 6.97ppm (d, phenyl proton), 5.21ppm (m, -OCH (CH)₃)CONH-)，4.93ppm(m，-OCH₂COO-)，3.53ppm(s，-CH₂CH₂O-)，1.49(d，-OCH(CH₃)CONH-)ppm。

Synthesis of PLGA-PEG-Cy5.5(PP-Cy5.5)

NH₂-synthesis of PEG-Cy5.5. Reacting NH₂PEG-thiol (0.014g, 0.06mmol) and sulfo-cyanine 5.5 MalaylImine (5mg, 0.072mmol) was dissolved in PBS (pH 7.0, 2mL) while drum N₂And removing oxygen. The solution was stirred and protected from light at room temperature for 48 hours. NH was obtained by dialysis with water (MW ═ 2Kda) until no absorption at 673nm of the dialysate occurred₂PEG-Cy5.5. Finally, NH was obtained by lyophilization₂PEG-Cy5.5. Yield: 0.018g (99%).

Synthesis of PLGA-PEG-Cy5.5 (PP-Cy5.5). PLGA (0.132g) was dissolved in dry DCM (1.5 mL). EDC. HCl (6mg, 0.03mmol) in 0.5mL of anhydrous DCM and NHS (3.7mg, 0.03mmol) in 10. mu.L of DMSO were added successively. The solution was stirred at room temperature for 2 hours. PLGA-NHS was obtained by precipitation in cold methanol/ether (50/50v/v), further washed twice and dried by rotary evaporation. PLGA-NHS was dissolved in dry DCM (2mL) and NH was then added₂PEG-Cy5.5(0.018g) is dissolved in the solution. DIPEA (7. mu.L) was added, and the mixture was stirred at room temperature for 48 hours. PLGA-PEG-Cy5.5(PP-Cy5.5) was obtained by precipitation in methanol/ether (50/50v/v) and washed 3 times with methanol/ether and finally dried in vacuo to remove the solvent. Yield: 0.106g (72%).

Synthesis of PLGA-PEG (PP)

PLGA (0.2g, 5. mu. mol) was dissolved in dry DCM (1.5 mL). EDC. HCl in 0.5mL of anhydrous DCM (10mg, 0.05mmol) and NHS in 10. mu.L of DMSO (5.7mg, 0.05mmol) were added sequentially. The solution was stirred at room temperature for 2 hours. PLGA-NHS was obtained by precipitation in cold methanol/ether (50/50v/v), further washed twice and dried by rotary evaporation. PLGA-NHS was dissolved in dry DCM (2mL) and NH was then added₂PEG-OH (0.018g, 6. mu. mol) was dissolved in this solution. DIPEA (10. mu.L) was added, and the mixture was stirred at room temperature for 24 hours. PLGA-PEG (PPG) was obtained by precipitation in methanol/diethyl ether (50/50v/v) and washed three times with methanol/diethyl ether and finally dried in vacuo to remove the solvent. Yield: 0.2g (90%).¹H NMR(CDCl₃，400MHz)：5.21ppm(m，-OCH(CH₃)CONH-)，4.82ppm(m，-OCH₂COO-)，3.64ppm(s，-CH₂CH₂O-)，1.57(d，-OCH(CH₃)CONH-)ppm。

Synthesis of cationic lipid (G0-C14)

The cationic lipoid compound G0-C14 is synthesized by the ring-opening reaction of PAMAM dendrimer G0 with 1,2 epoxy tetradecane according to the method described previously [28 ]. Briefly, 1, 2-epoxytetradecane and PAMAM dendrimer G0 were reacted in a 5: 1, by adding a sub-stoichiometric proportion of 1,2 epoxytetradecane, a product with three fewer tails than a given amine monomer is obtained. The mixture was stirred vigorously at 90 ℃ for 2 days.

Preparation of NPs

All NPs were prepared by nanoprecipitation method, PPM NPs were prepared as follows. 5mg PPM material containing 10% (mass fraction) PP-Cy5.5 was dissolved in DMSO (1mL) and added dropwise to an aqueous PVA solution (10mL, 0.1% w/v) stirred at 500 rpm. After the dropwise addition, the mixture was continuously stirred for 30 min. The mixture was then transferred to an Amicon Ultra-4 centrifugal filter (Millipore, Billerica, Mass.) with a molecular weight cut-off of 100kDa and washed 3 times to remove organic solvents and free molecules. NPs were diluted 20-fold in water and then measured for size using DLS. The fluorescent-labeled NPs containing the polymer PP are prepared by blending PP-Cy5.5 and PP, and the fluorescent-labeled PD NPs are prepared by blending PD-Cy5.5 and PD.

Macrophage culture and NPs uptake assay

RAW264.7 cells were treated in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin (P/S) at 37 ℃ with 5% CO₂Is cultured in a humid environment. Fluorochrome (i.e., PD-Cy5.5 or PP-Cy5.5) doped NPs at concentrations of 0, 0.45, 0.9, 1.6 and 2.8mg/mL were then added to the wells, incubated for 4h, and then replaced with fresh complete medium. After further incubation for 12h, the cells were washed 3 times with PBS and blown out of the well plate. Cells suspended in PBS were analyzed by flow cytometry to assess the uptake of NPs by the cells.

Cytotoxicity of NPs

For NPs cytotoxicity assays, Raw264.7 cells were used1×10⁴The cells/well density were seeded in 96-well cell culture plates and cultured for 24 h. NP dispersions were diluted with PBS and added to the wells at concentrations of 0, 0.9, 1.6, and 2.8mg/mL, respectively. After another 24h incubation, cell viability was analyzed by the CCK-8 assay. Briefly, the medium was removed and the cells were gently washed twice with PBS. Add 10. mu.L of CCK-8 solution to each well of the plate and incubate for 2 h. Absorbance at 450nm was measured using a microplate reader.

Nucleic acid complexing ability of G0-C14 and stability thereof in organic solvent

To evaluate the nucleic acid complexing ability of G0-C14 and its stability in organic solvents (DMF), naked nucleic acid or nucleic acid complexed with G0-C14 (weight ratio of 0.1 to 40) was incubated for 10min with or without DMF. For nucleic acid samples in DMF, electrophoresis does not require extraction of nucleic acid from DMF into aqueous solution. The volume of the sample was then adjusted with a dye and the sample was run on an E-Gel 1% agarose (Invitrogen) Gel at 100V for 20 min. Ambion Millennium marker formamide (Thermo Fisher Scientific) was used as a molecular weight standard. Finally, the gel was imaged under uv and the bands analyzed.

Preparation of nucleic acid NPs

We used a robust self-assembly method to prepare polymer-lipid hybridized NPs encapsulating nucleic acids (GFP DNA or EGFP mRNA). PP, PPM, PD and G0-C14 were each present in 2mg ml^-1Is dissolved in DMF. PP NPs without targeting moiety were prepared as follows. PP (300. mu.g in 150. mu.L) and G0-C14 (300. mu.g in 150. mu.L) were mixed in a glass vial at a ratio of 1: 1 by weight ratio. Nucleic acid (10. mu.g, concentration 1mg mL) in an aqueous solution was added^-1) Mixed into a PP/G0-C14 organic solution (nucleic acid: PP: the weight ratio of G0-C14 is 1: 30: 30) forming a cationic lipid/nucleic acid nanocomplex. This solution was then precipitated dropwise into 10mL of an aqueous PVA solution (2.5 mgml in DNase/RNase-free Hypure water, concentration)^-1). After nanoprecipitation, NPs were formed immediately and stirred at 500rpm for 20min to stabilize them. The NPs were then washed 3 times with ice cold Hypure water using Amicon tubes (molecular weight cut-off 100 kDa; Millipore), organic solvents and free compounds were removed, and finallyConcentrate to 1mL PBS solution. NPs can be used immediately or stored at-80 ℃ for later in vitro studies. As described above, NPs loaded with mRNA or pDNA were subjected to gel electrophoresis to examine whether or not the unencapsulated nucleic acids were leached out. According to the above protocol, PP is partially or completely replaced by PPM or PD to obtain NPs with targeting moieties, detailed information is given in Table S1&S2。

Release of mRNA from NPs

To analyze the release profile of NPs, we first encapsulated Cy 5-labeled EGFP mRNA (5-moUTP, APExBIO Technology LLC, texas) into NPs. The suspension of NPs in PBS was aliquoted (100. mu.L) into several semipermeable microdialysis tubes (molecular weight cut-off 100 kDa; Pierce); it was dialyzed against 2L of PBS (pH 7.4) which was frequently replaced at 37 ℃ with gentle stirring. At predetermined time points, five aliquots of each NP (n-5) were removed and the amount of encapsulated mRNA in NPs was determined using a standard curve correlating fluorescence and Cy5 mRNA concentration. Fluorescence intensity was measured by a multimode microplate reader (excitation/emission 650/670 nm; TECAN, Inc.). The amount of released mRNA was calculated to obtain a cumulative release curve.

Cell culture

The RAW264.7 cell line purchased from American Type Culture Collection (ATCC) was studied in vitro. Cells were cultured in DMEM cell culture medium and supplemented with 10% FBS inactivated at 56 ℃ for 30min and 1% P/S. Cell culture and all biological experiments were performed in a cell incubator at 37 ℃ under 5% CO 2.

In vitro transfection Activity of nucleic acid NPs

RAW264.7 cells were plated at 1X 10 per well⁵The density of individual cells was seeded on 24-well plates, allowed to attach and cultured for 24 h. Different nucleic acid concentrations (0.125, 0.250, 0.500 and 1.000. mu.g mL) in DMEM^-1) The cells were transfected with the nucleic acid NPs of (1) for 4h, then replaced with fresh complete medium, and incubated for another 24h to check the transfection efficiency. To measure transfection efficiency, cells were harvested and washed twice and resuspended in PBS and GFP expression was measured using flow cytometry. Data were analyzed using Flowjo software and expressed as percentage of GFP positive cells and Mean Fluorescence Intensity (MFI).

Statistical analysis

Data are presented as mean ± Standard Deviation (SD). Statistical analysis bilateral t-tests were performed using GraphPad Prism 8.0 software. P values less than 0.05 are considered statistically significant.

Example 1: synthesis and characterization of carbohydrate-conjugated PLGA or PLGA-PEG polymers

As shown in FIG. 1A, we use PD¹Peak c (4.49ppm, dextran terminal protons) and peak a (5.21ppm, -OCH (CH) in the H NMR spectrum₃) CONH-) to assess the number of PLGA chains per dextran. The number of PLGA chains grafted to the glucan can be controlled by adjusting the feed molar ratio (. gamma.) when PLGA/D-70-NH₂At 18.4, the average number of PLGA chains grafted per dextran was 20. GPC traces of PD show no residual unreacted PLGA, and the polymer has low polydispersity (. eta.)<1.2) (fig. 1D). PPM and PPG polymers were synthesized in a similar manner by acylation, amine deprotection and acylation in this order (scheme 2)&3). In the first step, the Boc (t-butyloxycarbonyl) was calculated by calculating the phenyl protons (7.5, 7.0ppm) and the t-butyl protons (1.37ppm, -C (CH)₃)₃) The integrated peak area of (A) gives a reaction rate of mannose or galactose grafted to PEG of 100% (FIG. S4)&S5). Second, Boc group was completely removed due to disappearance of 1.37ppm peak (FIG. S6). Finally, performing acylation reaction with PLGA to prepare PPM and PPG,¹the H NMR spectra are shown in FIGS. 1B and 1C, respectively. All reaction rates were above 80% by calculating the integrated area of peaks f and d.

Example 2: preparation and characterization of NPs

We used carbohydrate-modified polymers to design and prepare a series of NPs by nanoprecipitation (fig. 2A). Briefly, the polymer is dissolved in an organic solvent and added dropwise to deionized water. Two NPs were formulated, including NPs of the same composition (i.e., PPM, PPG, and PD) and a composition consisting of PPM and PPG in a weight ratio of 1: 1, to form a PPM/G NP. In addition, Cy5.5-labeled PLGA-PEG (PP-Cy5.5) was incorporated into NPs during formulation for subsequent flow cytometry to detect phagocytosis of NPs by macrophages. The average hydrodynamic size of the resulting NPs was measured using Dynamic Light Scattering (DLS). We have found that the particle size depends on the type of organic solvent used and the polymer concentration. The particle size was larger when NP was formulated with acetone compared to DMSO, and an increase in polymer concentration also resulted in an increase in particle size. In addition, the size of PD NPs also depends on the number of PLGA chains grafted to the dextran. PLGA chains with average numbers of 10, 20 and 33 were grafted onto dextran, respectively, and the corresponding PD NPs had average sizes of 85nm, 147nm and 263nm, respectively. The particle size gradually increases with the addition of the hydrophobic central core PLGA, which constitutes the NP. Therefore, we prepared NPs using PD containing 20 PLGA chains per dextran to achieve size uniformity for all NPs (fig. 2B). All NPs have a size of 136 + -10.9 nm (mean + -SD) and a polydispersity index (PDI) of 0.2 or less. TEM images of uranyl acetate stained PPM NPs showed monodisperse and spherical morphology with particle size less than DLS due to syneresis (figure 2C).

Example 3: macrophage uptake of NPs

To investigate the effect of the type of sugar on the macrophage targeting ability of NPs, cy5.5 was added during the self-assembly process to prepare fluorescently labeled NPs with various carbohydrate moieties. These fluorescently labeled NPs were then incubated at certain concentrations for different periods of time than RAW264.7 macrophages. Cells without NP treatment served as a control. Cy5.5 positive cells were examined using flow cytometry to explore the effect of sugar type on NP macrophage targeting ability. As shown in the histogram (fig. 3), if more cells take up the fluorescently labeled NPs, the fluorescence peak shifts to the right compared to the control group. Treatment with PP NPs containing no carbohydrate fraction at a maximum concentration of 2.8mg/mL yielded only 34% Cy5.5 positive cells. Note that NPs with carbohydrate moieties, particularly PD NPs and PPM NPs, showed higher endocytosis efficiency than non-targeted PPNPs, even at low concentrations of 0.45mg/mL, suggesting that incorporation of dextran or mannose moieties can promote receptor-mediated internalization of NPs (figure 3). Replacement of PPM with PPG fraction resulted in decreased cellular uptake of NPs. The Cy5.5 positive signal increases significantly with increasing concentration of sugar-modified NPs, reaching 90% or more at concentrations of 1.6mg/mL or higher. NPs modified with dextran or mannose moieties were used in the following in vitro transfection experiments.

Example 4: in vitro cell viability of NPs

One promising non-viral vector should achieve both sufficient transfection efficiency and low cytotoxicity. NPs were developed using carbohydrate-modified PLGA or PLGA-PEG synthesis, a biocompatible, biodegradable and safe administrable polymer and were approved by the FDA (food and drug administration) and EMA (european medicines administration) in the united states. The cytotoxicity of different concentrations of NPs was analyzed by CCK-8 in Raw264.7 cells in vitro (FIG. 4). The data show that at very high NPs concentrations of 2.8mg/mL (well above the concentration required for gene transfection), neither non-targeted PP NPs nor sugar-modified NPs significantly affected cell viability. These results indicate that both PP NPs and the sugar-modified NPs tested can be used as safe vectors for gene delivery in vivo.

Example 5: in vitro transfection Activity

Hybrid NPs are prepared by a nanoprecipitation method using cationic lipid compound G0-C14 and sugar-modified PLGA or PLGA-PEG polymer. FIG. 5A is a preparation of PPM/G0-C14 NPs loaded with pDNA. G0-C14 were used for gene recombination and the polymer was used to make a stable NP core. In addition, the polymer/G0-C14 layer can protect and capture the nucleic acid payload in the NP to control gene release. The Cy 5-labeled EGFP mRNA was first encapsulated into NPs, and then the amount of encapsulated mRNA was determined by fluorescence measurement according to a standard curve for Cy5 mRNA. The results show that PPM/G0-C14 NPs are able to encapsulate more than 95% of the initial mRNA. To measure mRNA release kinetics, PPM/G0-C14 NP samples loaded with Cy5 mRNA were dialyzed against 2L of frequently changed PBS (pH 7.4) at 37 ℃ to mimic physiological conditions. The release profile of Cy5 mRNA was measured using fluorescence spectrophotometry and showed that about 50% of total mRNA was released from NPs within the first 30h and then reached a maximum of 89% within 8d (FIG. S7). The above results show that the synthetic polymer/G0-C14 NPs are densely loaded with nucleic acids and achieve sustained release.

The ability of the G0-C14 vector to complex mRNA and pDNA was evaluated by electrophoretic blocking assay at different weight ratios. The gene-complexing ability of G0-C14 was evaluated using EGFP mRNA and GFP pDNA as model mRNA and pDNA. As shown in FIG. 5B, the migration intensity of the free mRNA or pDNA bands gradually decreased with increasing weight ratio (from 0.1/1 to 30/1 or 0.1/1 to 4/1, respectively), indicating that G0-C14 has stronger gene compression ability. G0-C14 efficiently concentrated EGFP mRNA and GFP pDNA, resulting in complete blockade of EGFP mRNA and pDNA at a weight ratio of 5 or higher. To achieve higher encapsulation efficiency after multiple washes during NP production, a weight ratio of 30 was chosen to formulate NPs for subsequent in vitro transfection experiments.

We incorporate sugar-modified polymers (PPM or PD) into PP at different ratios to completely replace PP, thereby preparing gene-loaded NPs with gradient targeting. For example, PP/PPM (4/1) NPs are made from PP and PPM polymers blended at 4/1 mass ratios. The hydrodynamic size of the NPs measured was about 200nm, with a narrow distribution, consistent with the DLS characteristics (tables S1 and S2). The average surface charge of the NPs is nearly neutral (0. + -. 0.57mV) because the NPs have an outer lipid-PEG-mannose or lipid-dextran shell. The NPs were found to be intact and spherical by Transmission Electron Microscopy (TEM), with a dehydrated size of about 50nm (fig. 5C). We also studied the stability of NPs, exemplified by PP/PPM (4/1) NP, and found that the size and Zeta potential of NPs did not change significantly after incubation at 37 ℃ for 24 hours (Table S3).

To further investigate the effect of sugar content on macrophage targeting ability of NPs, cy5.5-labeled PP or PD polymers (PP-cy5.5 or PD-cy5.5) were incorporated into the formulation process to prepare fluorescently labeled NPs with varying content of mannose or glucan moieties as described above. Binding and uptake of sugar-modified NPs and non-targeted PP NPs produced within RAW264.7 macrophages was quantitatively assessed by flow cytometry for detection of cy5.5 positive cells and MFI. As shown in fig. 6, the NPs produced showed strong cellular internalization in macrophages and increased dose-dependently. The percentage of Cy5.5 positive cells was significantly increased with sugar-modified NPs at doses ranging from 3.75. mu.g/mL to 15. mu.g/mL compared to non-targeted PP NPs (FIG. 6A). Under the NPs dosage of more than or equal to 30 mug/mL, the enhancement effect is moderate because the percentage of Cy5.5 positive cells reaches more than 90 percent. MFI data indicate that the phagocytic efficiency of NPs modified with sugars, in particular PP/PPM (4/1) NPs, PPM NPs and PP/PD (4/1) NPs, was significantly higher than that of non-targeted PP NPs at all doses tested (FIG. 6B). However, higher PD content in NPs resulted in reduced fluorescence intensity, suggesting that there is optimal carbohydrate density for specific interaction of PD-modified NPs with macrophages.

Then, we evaluated the in vitro gene transfection efficiency of NPs modified with different amounts of mannose or dextran moieties in Raw264.7 cells using EGFP mRNA and GFP pDNA as reporter genes. NPs encapsulating EGFP mRNA and GFP pDNA mediated high efficiency transfection in Raw264.7 cells, showing that there was a dose-dependent correlation of increased GFP expression, with an increase in gene concentration (from 0.125 to 1.0 μ g mL "1) (fig. 7). It is important to note that targeting PPM and PP/PD NPs mediated mRNA transfection efficiency at different concentrations was significantly higher than non-targeting NPs (FIGS. 7A and B). This is consistent with the above studies, i.e., PPM NPs and PP/PD (4/1) NPs show higher endocytosis efficiency. However, there was no clear correlation between endocytosis efficiency of PPM and PP/PD NPs and pDNA transfection efficiency at concentrations of 0.125. mu.g/mL and 0.250. mu.g/mL (FIGS. 7C and D). This may be due to the pattern of gene expression. First, macrophages will phagocytose gene-loaded hybrid NPs. Once NPs escape from the lysosome and release the gene payload into the cytoplasm, the information encoded by the released mRNA is translated into the protein of interest, but pDNA needs to enter further into the nucleus and be transcribed to express the protein. In addition to cellular internalization, nuclear entry would be another obstacle that must be overcome for successful delivery of pDNA.

Discussion of the related Art

Macrophages have a strong phagocytic capacity due to their main role in eliminating cell debris, pathogens, and foreign substances, and can internalize micro/nanoparticles indiscriminately. In addition to the above passive targeting, a number of targeting ligands for macrophage active targeting, such as mannose, folate, legumain, transferrin, and M2 macrophage targeting peptide (M2pep), etc., have been studied in the preclinical field. Among them, the biocompatibility and specific receptor recognition ability of saccharides make them potential ligands for macrophage-targeted drugs and gene delivery. Reportedly correspond toCompared with the carrier which is not modified by the saccharides, the mannosylated lipid complex, the poly (L-lysine), the cationic solid lipid NPs, the chitosan NPs, the polyamide amide and the polyethyleneimine can obviously improve the binding capacity with the macrophages. In addition to the mannose receptor (MMR, cluster of differentiation 206, CD206), other carbohydrate receptors are also expressed on the macrophage surface, such as macrophage galactose binding lectin (MGL, CD 301). Mannose and galactose have been used to target macrophages in conjunction with drugs or vaccines to modulate and enhance immune responses. Mannose modified for lymph imaging

Tc 99m telmancept agent is the first and only macrophage-targeting lymphographic agent approved by FDA and can specifically target the CD206 mannose receptor expressed on activated macrophages. In addition to monosaccharides, dextran has also been shown to be effective in targeting fat macrophages in obese mice. However, most macrophage-targeting vectors are used as imaging agents or for the delivery of small molecule drugs, but their use for delivery of biological macromolecules, particularly nucleic acids (such as mRMA or pDNA), has been less reported.

Heretofore, the present inventors have developed a hybrid NP platform by self-assembling biodegradable PLGA-PEG diblock copolymers and self-synthesized cationic lipid molecules G0-C14. G0-C14 consists of a cationic head group that can effectively capture therapeutic nucleic acids through electrostatic interactions and a flexible hydrophobic tail that can self-assemble with PLGA-PEG to form NPs. Cationic head groups include a number of primary, secondary and tertiary amine groups, which have strong proton buffering capacity, induce a proton sponge effect, and efficiently release the load into the cytoplasm. The proton-sponge effect of G0-C14 has been demonstrated in previous work. The PLGA-PEG/lipid hybrid NP platform can synergistically deliver siRNA and a cisplatin prodrug, continuously release effective load and has a good treatment effect on tumor cells.

In the invention, the structure of the cationic lipid G0-C14 is successfully optimized by adjusting the molar ratio of the dendritic macromolecule to the 14 carbon tail so as to promote the capture and transmission of macromolecular nucleic acid (namely mRNA or pDNA). A series of polymer/lipid hybrid NPs are prepared by utilizing different carbohydrate-modified PLGA or PLGA-PEG polymers to encapsulate G0-C14/mRNA or G0-C14/pDNA complexes through a nano precipitation method. NPs were prepared using various carbohydrates including mannose, galactose, dextran, and PLGA-PEG polymers constructed from a mixture of mannose and galactose. And (3) detecting the uptake of the fluorescence-labeled NPs by the macrophages by using flow cytometry. The cytotoxicity was determined using CCK-8. NP-mediated gene transfection in macrophages was studied using EGFP mRNA and GFP pDNA as reporter genes. The effect of different carbohydrate and component contents on targeting ability and transfection efficiency of engineered NPs was also investigated.

Macrophages play a key role in the pathological process of various inflammatory diseases such as diabetes, obesity, cancer, pulmonary fibrosis, cardiovascular diseases and the like. Macrophage targeted gene therapy, as a reversible method, can reverse potential pathological damage of inflammatory parts, and has a wide prospect, so that the development of a macrophage targeted gene vector capable of safely and effectively delivering therapeutic genes to an inflammatory microenvironment is urgently needed.

In this study, the progress and study of the targeting of gene delivery to macrophages by controlled release NPs modified with different sugars was demonstrated. The results show that the incorporation of carbohydrate moieties, especially mannose and dextran, significantly improves the active targeting of macrophages and the gene transfection efficiency of NPs. Furthermore, gene transfection efficiency depends on the type and content of carbohydrate moieties. Notably, the major components of NPs are FDA-approved materials, making NPs an attractive gene vector system with clinical transformation potential. This work provides a potential technological platform for the delivery of biomacromolecules and therapeutic genes at the site of macrophage inflammation, which is encouraging toward the clinical transformation of targeted gene therapy for the treatment of macrophage-mediated inflammatory diseases.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

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<120> components of macrophage targeting vector system, preparation method and application thereof in drug and nucleic acid delivery

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Claims

1. A nanoparticle for drug and gene delivery targeting macrophages, wherein the nanoparticle comprises:

(i) a polymer segment for forming a matrix of the nanoparticle;

wherein the targeting element is selected from the group consisting of:

(Y4) any combination of the above Y1 to Y3;

with the proviso that,

2. The nanoparticle according to claim 1, wherein said nanoparticle further comprises (v) a loaded drug, said loaded drug being distributed or entrapped within the matrix of said nanoparticle.

3. The nanoparticle according to claim 1 or 2, wherein the nanoparticle further comprises (vi) lipid molecules distributed or encapsulated on the outer surface or inside of the nanoparticle.

4. The nanoparticle according to claim 1, wherein the nanoparticle comprises a complex of a plurality of nanoparticle units having a structure selected from the group consisting of: formula I, formula II or formula III or formula IV;

Z1-Z2-Z3a (formula I)

Z1-Z2-Z3b (formula II)

(Z1) m-Z4- (Z3) n (formula III)

(Z1-Z2-Z3) p-Z4 (formula IV)

Wherein the content of the first and second substances,

z1 is a polymer segment;

z2 is a linking element;

z3b is a developer;

z3 can be Z3a, Z3b, or a combination of Z3a and Z3b in any ratio;

z4 is macrophage targeting glycan component Y3;

m is an integer of 1 or more (preferably 1 to 432);

n is an integer of 0 or more (preferably 0, or 1 to 432);

p is an integer of 1 or more (preferably 1 to 432)

"-" denotes a chemical bond.

5. The nanoparticle of claim 4, wherein said nanoparticle unit further comprises a drug distributed or encapsulated on the interior or exterior surface of said nanoparticle unit.

6. The nanoparticle according to claim 5, wherein said nanoparticle unit further comprises a lipid molecule complexed with said drug, distributed or encapsulated within or on the outer surface of said nanoparticle.

7. A pharmaceutical composition, comprising:

(i) the nanoparticle of claim 1, wherein the nanoparticle comprises a loaded drug distributed or entrapped on or within the outer surface of the nanoparticle; and

(ii) a pharmaceutically acceptable carrier.

8. A method of preparing nanoparticles according to claim 1, comprising the steps of:

Z1-Z2-Z3a (formula I)

Z1-Z2-Z3b (formula II)

(Z1) m-Z4- (Z3) n (formula III)

(Z1-Z2-Z3) p-Z4 (formula IV)

Wherein the content of the first and second substances,

z1 is a polymer segment;

z2 is a linking element;

z3b is a developer;

z3 can be Z3a, Z3b, or a combination of Z3a and Z3b in any ratio;

z4 is macrophage targeting glycan component Y3;

m is an integer of 1 or more (preferably 1 to 432);

n is an integer of 0 or more (preferably 0, or 1 to 432);

p is an integer of 1 or more (preferably 1 to 432)

"-" denotes a chemical bond; and

(ii) mixing the nanoparticle units to obtain the nanoparticle of claim 1.

9. The method of claim 8, wherein the nanoparticle unit comprises a drug distributed or encapsulated on the interior or exterior surface of the nanoparticle unit.

10. Use of a nanoparticle according to claim 1 for the preparation of a medicament for targeting macrophages or for the treatment of a disease associated with macrophage inflammation.