CN-122005774-A - Inhalable targeting gold nanoparticles for accelerating pulmonary delivery and treating pulmonary inflammation

Abstract

The present invention provides a method of preparing inhalable targeted gold nanoparticles for accelerating pulmonary delivery and treating pulmonary inflammation. The method comprises the steps of preparing plasmids, carrying out plasmid transformation, carrying out protein expression induction, carrying out protein purification, and preparing spike RBD-combined gold nanoparticles (Au@PEG-RBD NP). Nanoparticles (NPs) in air accumulate more readily in the lungs with lipopolysaccharide-induced Acute Respiratory Distress Syndrome (ARDS) than in healthy lungs and enter alveolar epithelial cells that express Angiotensin Converting Enzyme (ACE) 2 and L-SIGN, both Spike (Spike) receptors activated in ARDS hamsters. The Nanoparticle (NP) is more effective than corticosteroids in treating tissue injury, oxidative stress and inflammation, and does not leave gold or cause toxicity in major organs after inhalation for one year. The gold core inhibits p38 alpha mitogen-activated protein kinase and polo-like kinase 3. Similar efficacy was observed in hamsters with hydrochloric acid induced ARDS. Such self-therapeutic Nanoparticles (NPs), when combined with biomimetic (bioinspiration) and non-invasive delivery, provide a safe, efficient and targeted treatment for pulmonary inflammation.

Inventors

• CAI ZONGHENG
• LIU SHAORUI

Assignees

• 香港中文大学

Dates

Publication Date

20260512

Application Date

20251112

Priority Date

20241112

Claims (20)

1. 1. A method of preparing inhalable targeted gold nanoparticles for accelerating pulmonary delivery and treating pulmonary inflammation, the method comprising: Preparing a plasmid; carrying out plasmid transformation; carrying out protein expression induction; Protein purification and Spike RBD-conjugated gold nanoparticles (Au@PEG-RBDNP) were prepared.
2. 2. The method of claim 1, wherein the preparing a plasmid comprises: DH5 Mixing competent cells with plasmid; incubating the mixture on ice; subjecting the mixture to heat shock; Incubating the mixture on ice; Adding a preheated lysozyme broth to said mixture, and Cells in the mixture were incubated with orbital shaking.
3. 3. The method of claim 2, further comprising spreading the transformed cells onto LB agar plates containing ampicillin and incubating the cells.
4. 4. The method of claim 3, further comprising selecting a population of the cells and growing the selected population in LB/ampicillin under shaking.
5. 5. The method of claim 4, further comprising harvesting the cells by centrifugation and purifying the plasmid to obtain a plasmid encoding His-tagged RBD (pET 11a-RBD-8 xHis).
6. 6. The method of claim 1, wherein performing plasmid transformation comprises: thawing on ice was previously performed at-80 Lower stored competent cells; Adding a plasmid to the competent cells to obtain a mixture; holding cells of the mixture on ice; heat-shock the cells in the mixture in a water bath and incubating the cells again on ice, and Fresh LB medium was added and the mixture was shaken.
7. 7. The method of claim 6, further comprising centrifuging the cells of the mixture and discarding the supernatant.
8. 8. The method of claim 7, further comprising re-suspending remaining pelleted cells.
9. 9. The method of claim 8, further comprising inoculating bacteria onto LB agar plates with antibiotics and incubating the bacteria.
10. 10. The method of claim 1, wherein said performing protein expression induction comprises: transforming an expression strain Origami B cell pre-transformed with a chaperone plasmid pG-KJE by using an RBD expression plasmid pET11 a-RBD; culturing the resultant on LB agar plates; selecting a single colony from the resultant; Adding the community to fresh LB medium with antibiotics; adding an overnight culture to the fresh LB medium, and The resultant was incubated until OD 600 reached about 0.5.
11. 11. The method of claim 10, further comprising inducing by adding an induction buffer comprising isopropyl β -D-1-thiogalactopyranoside and L-arabinose.
12. 12. The method of claim 11, further comprising harvesting the cells by centrifugation for purification or at-80 And (5) storing the mixture under the condition.
13. 13. The method of claim 1, wherein the performing protein purification comprises: resuspending Origami B cells in a protein purification buffer and lysing the cells by an ultrasonic processor to produce a lysate; centrifuging the lysate; Collecting the supernatant of the lysate; filtering the supernatant with a syringe filter; incubating the supernatant with Ni-NTA resin to obtain a lysate-resin mixture, and The lysate-resin mixture was transferred to a blank gravity chromatography column along with the resin.
14. 14. The method of claim 13, further comprising discarding liquid flowing through the lysate-resin mixture and adding a wash buffer to elute non-specific binding proteins.
15. 15. The method of claim 14, further comprising eluting the RBD protein product by adding an elution buffer and dialyzing it against a storage buffer.
16. 16. The method of claim 15, further comprising determining the purity and concentration of the protein product by Sodium Dodecyl Sulfate (SDS) -polyacrylamide gel electrophoresis (PAGE) and Bradford assay, respectively.
17. 17. The method of claim 1, wherein the preparing spike RBD-bound gold nanoparticles (au@peg-RBD NP) comprises: Citrate capped gold nanoparticles (cit-AuNP) were synthesized with a diameter of about 20 nm; after boiling HAuCl 4 , sodium citrate was added with vigorous stirring and the mixture was kept boiling; Cooling the resultant to obtain cit-AuNP, and Newly dissolved thiol (HS) -PEG 20k -methoxy or HS-PEG 20k -nitrilotriacetic acid (NTA) (Biochempeg) was added to the cit-AuNP solution at a molar ratio of 1:1 at a total concentration of 5 PEG molecules per nm 2 nanoparticle surface to obtain the resulting mixture.
18. 18. The method of claim 17, further comprising stirring the resulting mixture, adding NiCl 2 , and then stirring to obtain au@peg-NTA-Ni 2+ nanoparticles (au@peg-NTA-Ni 2+ NP).
19. 19. The method of claim 18, further comprising adding the His-tagged RBD protein to an au@peg-NTA-Ni 2 + NP solution and agitating to obtain au@peg-RBD nanoparticles (au@peg-RBD NP).
20. 20. The method of claim 19, further comprising dialyzing the obtained au@peg-RBD NP against Nanopure ultrapure water by centrifugation.

Description

Inhalable targeting gold nanoparticles for accelerating pulmonary delivery and treating pulmonary inflammation Cross Reference to Related Applications The application claims the benefit of U.S. provisional application Ser. No. 63/71919, filed 11/12 of 2024, the entire contents of which, including any figures, tables, or graphics, are incorporated herein by reference. Background Acute Respiratory Distress Syndrome (ARDS) is the leading cause of death, with about 10% of intensive care patients suffering from ARDS and a global mortality rate of 40% [1]. Rupture of the air-blood barrier (ABB) is a direct pathophysiological cause [2] of ARDS, leading to leakage of biological fluids into lung tissue and disruption of oxygen diffusion into the blood. Severe inflammatory symptoms may occur [3] within 12 hours of exposure to bacterial or chemical stimuli. Thus, timely management will improve the treatment outcome [4]. However, conventional treatments require 5-7 days to reach clinical response and do not target disease causes [5,6], such as invasive mechanical ventilation (INVASIVE MECHANICAL ventilation) and anti-inflammatory drugs [7] with side effects. Nanoparticles (NPs) are being investigated for ARDS management [8]. Due to the high vascularization of the lung, the past lung nanomedicines were mostly delivered to the lung endothelium by intravenous injection (see tables 1 and 2). However, where carriers, drugs and targeting ligands are included, they are generally large and are readily cleared by the liver, which may lead to systemic toxicity. Lung-specific delivery is possible, but requires specialized lipid engineering (specialized LIPID ENGINEERING) [9]. Importantly, intravenous delivery does not guarantee delivery to the lung epithelium, which is a key structural component of ABB protection of the lung [10], regulation of ARDS-related immune response [11], and regulation of tissue repair [12]. Other Nanoparticles (NPs) were delivered locally to the lung epithelium using intratracheal injection (INTRATRACHEAL INSTILLATION) (table 2). However, instillation may raise airway pressure and cause cardiac arrest [13], with instilled NPs accumulating only in the stenotic tissue region (confined tissue region). Interestingly, inhalation provided rapid, non-invasive pulmonary delivery and broad tissue distribution [14], but until recently rare applications [15] were not available (table 1). It is hypothesized that inhalable NPs targeting the ARDS lung epithelium may support rapid, safe and effective treatment. Currently, there are four main types of treatments for ARDS, namely mechanical ventilation, corticosteroids, anti-inflammatory small molecule drugs and NP. Among them, mechanical ventilation is invasive to the trachea and only relieves symptoms. Furthermore, surgical opening of the trachea may lead to local infections, whereas inhaled NPs are not invasive. Corticosteroids have strong anti-inflammatory efficacy, although they have serious systemic side effects and cannot be used to treat ARDS specifically. Anti-inflammatory small molecule drugs (such as statins and ACE inhibitors) have been extensively tested (not yet approved) in ARDS human trials, but statins have no protective effect on ARDS, whereas ACE inhibitors have only a slight therapeutic effect. NP-based therapies are relatively safer than previously mentioned methods. There are several commonly used NPs for carrying ARDS drugs to the lung, such as liposomes, polymeric NPs and exosome-derived NPs (exosomes-DERIVED NP). However, liposomes require complex manufacturing processes, and the drug delivery efficiency of polymeric NPs is low, while exogenously derived NPs are more difficult to manufacture and yield is low. Furthermore, current NP delivery routes have limited efficacy, particularly in (1) intravenous (i.v.) or intraperitoneal (i.p.) and (2) intratracheal (i.t.). Intravenous or intraperitoneal injection provides systemic delivery. To maximize pulmonary delivery efficiency, targeting ligands are typically used, resulting in accumulation of most of the injected NPs in the liver and spleen. Intratracheal injection is invasive and risky, and may raise airway pressure and cause cardiac arrest. Furthermore, instilled NPs (in fluid) often accumulate in small tissue areas due to inertial impaction. Disclosure of Invention There remains a need in the art for improved designs and techniques for nanoparticle-based drug delivery methods that target the lungs. According to one embodiment of the present invention, a method is provided for preparing inhalable targeted gold nanoparticles for accelerating pulmonary delivery and treating pulmonary inflammation. The method comprises the steps of preparing plasmids, carrying out plasmid transformation, carrying out protein expression induction, carrying out protein purification, and preparing spike RBD-combined gold nano-particles (spike RBD-conjugated gold nanoparticles) (Au@PEG-RBD NP). Preparing plasmids include