AU2023263453B1 - A polypeptide carrier for delivering nucleic acid drugs, a nucleic acid drug for treating tumors, and preparation method thereof - Google Patents
A polypeptide carrier for delivering nucleic acid drugs, a nucleic acid drug for treating tumors, and preparation method thereof Download PDFInfo
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/5176—Compounds of unknown constitution, e.g. material from plants or animals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5192—Processes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The present disclosure relates to a polypeptide carrier for delivering nucleic acid
drugs, with amino acid composition as shown in SEQ ID NO. 3. The present disclosure
also provides an application of the polypeptide carrier and the corresponding nucleic
acid drugs for treating tumors, as well as the preparation method thereof. The MMP2
enzyme-responsive peptide designed in the present disclosure can effectively bind to
miRNA mimics, and RBCM endows the nanoparticles with a stealth function, thereby
improving their circulation life, immune evasion and biosafety. After the MMP2
enzyme-responsive biomimetic nanoparticles are absorbed by lung cancer cells, when
a large amount of MMP2 exists in lung cancer cells, miR-126-3p mimics are released,
effectively inducing cancer cell apoptosis. The nucleic acid drug for treating tumors
disclosed in the present disclosure provides a novel strategy for lung cancer treatment
with great application prospects.
Description
Technical Field
[0001] The present disclosure relates to the technical field of biomedicine, and in
particular, relates to MMP2 enzyme-responsive polypeptide carriers for delivering
nucleic acid drugs, nucleic acid drugs for treating tumors, and preparation method
thereof.
Background
[0002] Lung cancer is one of the deadliest cancers in the world, and conventional
treatments such as chemotherapy, radiotherapy, and surgical resection have limited
efficacy and serious side effects. At present, gene therapy has become an innovative
and powerful means of treating challenging diseases such as cancer. Research showns
that miR-126-3p has potential therapeutic effects in the treatment of lung cancer.
However, the strong negative charge of microRNAs (miRNAs) hinders their
internalization in the cell membrane, and rapid enzymatic digestion in the physiological
environment can hinder the delivery of miRNAs to the desired sites in the body. How
to safely and effectively transport miRNAs into the cytoplasm and release them remains
a major challenge in gene regulation and treatment. Currently, many carriers, including
viral and non-viral carriers, have been developed for delivering nucleic acids. However,
viral carriers have drawbacks such as low loading capacity,
inflammatory/immunogenic reactions, and difficulty in manufacturing on a large scale.
In recent years, non-viral carriers have received increasing attention on their application
in gene delivery due to their advantages such as convenient design, low cytotoxicity,
and intelligent responsiveness. In order to effectively deliver miRNAs into cancer cells,
drugs must overcome three transportation barriers: (1) avoiding being captured by the immune system and reaching the lesion, (2) being absorbed effectively by cancer cells, and (3) protecting and releasing therapeutic nucleic acids. Therefore, we designed an
MMP2 enzyme-responsive biomimetic nanoparticles wrapping microRNA to improve
the delivery of nucleic acid drugs to the target site.
[0003] Natural cell membranes-camouflaged nanoparticles are a new type of
biomimetic nanoparticles. The simple engineering of cell membranes endows
nanoparticles with highly complex functions. They possess both the unique functions
of cell membranes and the universality of nanomaterial synthesis, giving nanoparticles
wrapped in cell membranes significant advantages, such as prolonged circulation,
specific targeting, and immune evasion. At present, according to different needs,
biofilms, including tumor cells, stem cells, platelets, etc., have been widely studied in
the biomedical field. Red blood cells are oxygen transporters in our body, with a
lifespan of about 4 months and the ability to project many surface markers. Due to the
mediation of various proteins located on the red blood cell membranes, such as CD47,
which have self-recognition function and can prevent phagocytosis by immune cells,
thereby extending the circulation life of biomimetic nanoparticles. Polypeptide drug
carriers are one of the important non-viral carriers, and cell membrane-penetrating
peptides are rich in positively charged alkaline amino acid short peptides, which can
self-assemble with negatively charged nucleic acid drugs through electrostatic
interactions under physiological conditions to form non-covalent-bound nanoparticles.
While the overexpression of matrix metalloproteinase 2 (MMP2) in cancer cells can
specifically recognize and cleave peptide sequences Pro-Leu-Gly-Leu-Ala-Gly
[0004] Gene therapy is one of the important means of cancer treatment, but how to
safely, efficiently, and effectively deliver miRNAs into the body is an urgent problem that needs to be solved in current research. Currently, many carriers, including viral and non-viral carriers, have been developed for delivering nucleic acids. However, viral carriers have disadvantages such as low loading capacity, inflammatory/immunogenic reactions, and difficulty in manufacturing on a large scale. In recent years, non-viral carriers used for nucleic acid delivery mainly include organic polymers, inorganic nanoparticles, etc. Although these nucleic acid delivery carriers have been widely studied, they still have many shortcomings, such as poor delivery targeting of organic polymer, poor biodegradability of inorganic nanoparticles, and easy accumulation in the body leading to biological toxicity, etc.
[0005] The MMP2 enzyme-responsive peptide PLGLAG can be used to construct
environmentally responsive nanoparticles, thereby improving the release of miRNA to
lung cancer cells. However, the stability, biological targeting, and in vivo activity of
the PLGLAG sequence during delivery are not ideal.
Summary of the Disclosure
[0006] The present disclosure relates to a system and molecules for delivery of miR
126-3p mimics using MMP2 enzyme-responsive biomimetic nanoparticles.
[0007] This paragraph has been intentionally deleted.
[0008] In a first aspect, the present disclosure provides a polypeptide carrier for
delivering nucleic acid drugs, comprising an amino acid sequence as shown in SEQ ID
NO. 3.
[0009] In a second aspect, the present disclosure provides the use of the above
mentioned polypeptide carrier in the preparation of a drug for delivering a MicroRNA
drug.
[0010] In a third aspect, the present disclosure provides a nucleic acid drug for
treating tumors, wherein the drug comprises red blood cell membrane-wrapped nanoparticles formed by the polypeptide carrier and a tumor-targeting MicroRNA drug.
[0011] In some embodiments, the tumor is an MMP2 enzyme-responsive tumor.
[0012] In some embodiments, the tumor is lung cancer.
[0013] In some embodiments, the MicroRNA drug is miR-126-3p mimics.
[0014] In a fourth aspect, the present disclosure provides a preparation method of the
above-mentioned nucleic acid drug for treating tumors, including the following steps:
(1) obtaining red blood cell membrane;
(2) dissolving the polypeptide carrier in PBS to obtain a polypeptide carrier solution;
(3) dissolving a tumor-targeting MicroRNA drug in nuclease free water to obtain a
MicroRNA drug solution;
(4) mixing the polypeptide carrier solution and the nucleic acid drug solution (i.e.
MicroRNA drug solution) according to a mass ratio of the polypeptide carrier to the
tumor-targeting MicroRNA drug of 20:1 to 40:1, and letting it stand at room
temperature to allow the nucleic acid and the polypeptide carrier to form nanoparticles
through self-assembly of positive and negative charges;
(5) adding the red blood cell membranes to the obtained nanoparticles, mixing evenly,
and squeezing the prepared nanoparticles through gradient extrusion to obtain
nanoparticles with a uniform particle size to obtain a nucleic acid drug.
[0015] In some embodiments, the mass ratio of the polypeptide carrier to the tumor
targeting MicroRNA drug is 20:1 to 40:1, preferably 28:1 to 32:1.
[0016] In some embodiments, the mass ratio of the red blood cell membranes to the
nanoparticles is 5:1 to 30:1, preferably 8:1 to 12:1
[0017] In order to effectively deliver miRNAs into cancer cells, drugs must overcome
the following three transportation barriers: (1) avoiding being captured by the immune
system and reaching the lesion, (2) being effectively absorbed by cancer cells, and (3) protecting and releasing therapeutic nucleic acids. Therefore, the present disclosure constructs a system for efficient targeted delivery of miR-126-3p mimics using
MMP2 enzyme-responsive biomimetic nanoparticles. Firstly, a cationic peptide with
six arginine residues, 6R-PLGLAG-6R, is synthesized at both ends of the MMP2
enzyme-cleavable peptide PLGLAG. The cationic peptide can controllably load the
miRNA and release the miRNA under environmentally responsive conditions, which is
beneficial to the treatment of diseases. Secondly, the inventor of the present disclosure
screened out suitable preparation parameters based on accumulated experience. The
6R-PLGLAG-6R is combined with miR-126-3p mimics through electrostatic
adsorption, and is further modified with red blood cell membranes. The obtained
REMAIN has the smallest particle size and highest transfection efficiency, which is
conducive to the accumulation of nanoparticles into tumor tissue and to better delivery
of miR-126-3p. After the MMP2 enzyme-responsive biomimetic nanoparticles are
absorbed by lung cancer cells, and when a large amount of MMP2 exists in lung cancer
cells, 6R-PLGLAG-6R can be cleaved and decomposed by MMP2, and the miR-126
3p mimics are released, achieving controlled release and effectively inducing cancer
cell apoptosis. However, the application of such nanoparticles for the delivery of lung
cancer gene drugs has not yet been reported. The present disclosure explores and
develops a new strategy for the treatment of lung cancer with great prospects.
Brief Description of the Drawings
[0018] Fig. 1 Comparison of preparation conditions of nanomedicines MAIN and
[0019] Fig. 2 Characterization of nanomedicines MAIN and REMAIN.
[0020] Fig. 3 The changes in miR-126-3p expression levels in NCI-H299 cells
measured by RT-qPCR method, after the miR-126-3p was transfected with different polypeptide carriers and Lipo3000.
[0021] Fig. 4 In vivo anti-tumor effects of nanomedicines MAIN and REMAIN.
[0022] Fig. 5 HE staining results of major organ sections.
Detailed Description
[0023] In order to facilitate the understanding of the present disclosure, a more
comprehensive description about the present disclosure is given below. The present
disclosure can be implemented in many different forms, and is not limited to the
embodiments described herein. On the contrary, these embodiments are provided to
make the understanding of the disclosure more thorough and comprehensive.
[0024] Experimental methods without specifying specific conditions in the following
examples usually follow conventional conditions, such as those described in Sambrook
et al., Molecular Cloning:A LaboratoryManual, 2013 (New York: Cold Spring Harbor
Laboratory Press, 1989 ), or the conditions recommended by the manufacturers. The
various commonly used chemical reagents used in the embodiments are all
commercially available products.
[0025] Unless otherwise defined, all technical and scientific terms used herein have
the same meaning as commonly understood by the skilled in the art of the present
disclosure. The terms used in the description of the present disclosure are for describing
specific embodiments only and are not intended to limit the present disclosure. The
term "and/or" used in the present disclosure includes any and all combinations of one
or more associated listed items.
[0026] Before elaborating on the technical solutions of the present disclosure, the
terms used in this article are defined as follows:
[0027] The term "RBCM" refers to red blood cell membranes.
[0028] The term 'MAIN' refers to nanoparticles of MMP2 enzyme-responsive
PLGLAG- short peptides loaded with miR-126-3p mimics.
[0029] The term'REMAIN' refers to RBCM-modified MAIN.
[0030] The term 'REMAIN-NC' refers to RBCM-modified nanoparticles of MMP2
enzyme-responsive -PLGLAG- short peptides loaded with miRNA-NC mimics.
[0031] The term'PBS solution' refers to phosphate buffer solution.
[0032] The present disclosure designs polypeptides with good biocompatibility and
biodegradability for delivering miRNA for tumor treatment. The present disclosure
relates to a method and application of delivering miR-126-3p mimics based on red
blood cell membranes-modified MMP2 enzyme-responsive nanoparticles. The
modification of the red blood cell membranes endows nanoparticles with cell-like
functions, such as specific recognition, long-term blood circulation, and immune
evasion. The specific adhesion molecules based on on the red blood cell membranes,
the long blood circulation and cellular internalization of nanoparticles are significantly
improved. Red blood cell membranes with long blood circulation can be used as a
biomimetic delivery system for cancer-targeted gene therapy, inducing efficient
apoptosis of cancer cells and leading to significant tumor suppression.
[0033] In the present disclosure, a cationic peptide with 6 arginine residues (6R
PLGLAG-6R) is synthesized at both ends of the MMP2 enzyme-cleavable peptide
PLGLAG. the MMP2 enzyme cleaves both ends of the peptide PLGLAG to synthesize
a cationic peptide with six arginine residues (6R-PLGLAG-6R).Subsequently, the 6R
PLGLAG-6R binds to miR-126-3p mimics (MMP2 stimulated peptide/miRNA-126-3p,
MAIN) through electrostatic adsorption, and then MAIN is further modified with red
blood cell membranes (RBCM) forming (Red blood cell membranes/MMP2 stimulated
peptide/miRNA-126-3p, REMAIN).The obtained MMP2 enzyme-responsive peptide
can effectively bind to miRNA mimics, and the camouflage of RBCM endows the nanoparticles with a stealth function, thereby improving their circulation life, immune evasion, and biosafety. After REMAIN is absorbed by lung cancer cells and when a large amount of MMP2 exists in lung cancer cells, miR-126-3p mimics are released, effectively inducing cancer cell apoptosis.
[0034] The present disclosure will be further described in detail as below in
conjunction with specific embodiments.
[0035] Embodiment 1: Preparation of nanoparticles MAIN and REMANIN and
optimization of polypeptide sequences and ratio parameters
[0036] The present disclosure designs three polypeptides with different amino acid
sequences, namely 2, 4, and 6 arginines are connected to both ends of -PLGLA- peptide
segment, wherein, the amino acid sequences are respectively:
2R-PLGLAG-2R: RRPLGLAGRR (SEQ ID NO. 1) ,
4R-PLGLAG-4R: RRRRPLGLAGRRRR (SEQ ID NO. 2),
6R-PLGLAG-6R: RRRRRRPLGLAGRRRRRR (SEQ ID NO. 3)
[0037] In this embodiment, the preparation of the nucleic acid drug is as follows:
[0038] (1) Extraction of red blood cell membranes: red blood cells were extracted
from whole blood by centrifugation at 2000 rpm for 5 minutes, and lysed with red blood
cell lysate; further centrifuged at 14000 rpm at 4 °C for 15 minutes; redissolved the
precipitate in deionized water containing EDTA, sonicated for 3 minutes, and then
centrifuged at 14000 rpm at 4 °C for 15 minutes. Repeated the process twice. Finally,
the obtained red blood cell membranes were washed twice with deionized water
containing EDTA to remove hemoglobin.
[0039] (2) Three types of polypeptide carriers were respectively dissolved in PBS to
prepare g/L of polypeptide solution. miR-126-3p mimics was dissolved in
nuclease-free water to prepare 1 g/4L of nucleic acid solution. Added the nucleic acid solution to the peptide solution at mass ratios of nucleic acid: polypeptide=1:5, 1:10,
1:20, 1:30, 1:40, 1:50, 1:60, 1:90, and immediately performed vortex shaking for 1
minute to thoroughly mix the nucleic acid and polypeptide. Let stand at room
temperature for 15 minutes, allowing nucleic acids and polypeptides to form
nanoparticles MAIN through self-assemble of positive and negative charges.
[0040] (3) Added the RBCM obtained in step (1) to the MAIN obtained in step (2) at
mass ratios of MAIN: RBCM = 1:5, 1:10, 1:20, 1:30 respectively. The mixture was
gradient extruded through a squeeze film to obtain REMAIN with a uniform particle
size, i.e., the mixture was first extruded through a carbonate membrane with pore size
of 1 M using a squeeze film, and squeezed back and forth through the membrane
twenty times, and then used the same method to squeeze sequentially through carbonate
membranes with pore sizes of 400 nm, 200 nm, and 100 nm.
[0041] (4) After appropriately diluting the prepared nanomedicines, measured the
particle size and polydispersity index (PDI) of each group of nanoparticles using a
Malvern particle size analyzer.
[0042] (5) Added 10 L of each group of nanoparticles samples dropwise onto the
copper mesh of an electron microscope, let stand for 5 minutes, used absorbent paper
to absorb excess liquid along the edge of the copper mesh, and left the copper mesh at
room temperature overnight to dry. Took electron microscope photos of each group of
samples according to the operating procedures of transmission electron microscope to
obtain the morphological characteristics and particle size of each sample nanoparticles.
[0043] The results of the Malvern particle size analyzer showed that the overall
particle size of 6R-PLGLAG-6R was smaller than that of other peptide sequences (Fig.
1A), and the qRT-PCR results showed that the transfection efficiency of miR-126-3p
by 6R-PLGLAG-6R was higher than that of other peptide sequences, and was comparable to the transfection efficiency of the positive control group (Fig. IB). It was because the increase in the amount of arginine led to an increase in amino acids or imines in the polypeptide, increasing charge density, and improving the compactness of negatively charged miR-126-3p. Therefore, 6R-PLGLAG-6R was the most preferred polypeptide sequence. Higher transfection efficiency was beneficial for better delivery of miR-126-3p. The mass ratio of peptide to nucleic acid was 5:1 to 90:1, and the results of the Malvern particle size analyzer showed that the particle size and PDI were the smallest when the ratio was 30:1 (Fig. IC). Therefore, the optimal ratio of 6R
PLGLAG-6R: miR-126-3p is 30:1. The mass ratio of red blood cell membranes to
MAIN composite nanoparticles was 5:1 to 30:10, and the results of the Malvemparticle
size analyzer showed that the particle size tended to become saturated when the ratio
was 10:1 (Fig. ID). Therefore, the most preferred ratio of RBCM: MAIN was 10:1.
Smaller particle size facilitated the accumulation of nanoparticles into tumor tissue. In
the following embodiments, all MAIN and REMAIN were prepared in accordance with
the optimal proportions in this embodiment.
[0044] Embodiment 2: Characterization of Nanoparticles MAIN and REMAIN
[0045] MAIN and REMANIN nanoparticles were synthesized using the method of
Embodiment 1. Transmission electron microscopy (40K, 80K magnification) showed
that the MAIN nanoparticles were approximately spherical and uniform in size (Fig.
2A). The results of the Malvern particle size analyzer showed that the average particle
size of the MAIN nanoparticles was about 135.5 nm, with a PDI of 0.222. The size of
the nanoparticles was consistent with the results shown in the transmission electron
microscopy image (Fig. 2B). Transmission electron microscopy (40K, 80K
magnification) showed that the REMAIN nanoparticles were approximately spherical,
and the modification of the red blood cell membranes increased the size of the REMAIN by about 20 nm compared with the MAIN (Fig. 2C), with an average particle size of about 156.7 nm and a PDI of 0.239 (Fig. 2D). Equal amounts of total proteins of non small cell lung cancer NCI-H1299, normal lung epithelial cells BEAS-2B, and human umbilical cord mesenchymal stem cells hUV-MSCs were electrophoresed on 10%
SDS-PAGE (80V, 1.5 hours) and transferred to PVDF membrane (250 mA, 2 hours),
and then after incubation with the primary antibody probe MMP2 overnight, incubated
with the peroxidase-linked secondary antibody for 1 hour. Finally, imaging was
performed with an ultra-sensitive multifunctional imaging device. Western blotting
results showed that MMP2 was overexpressed in non-small cell lung cancer NCI
H1299 (Fig. 2F), and the environmental response characteristics of MMP2 enzyme
were favorable for drug-controlled release. Firstly, prepared REMAIN containing
FITC-miR-126-3p and loaded it into a dialysis membrane (MW=3500). The miR-126
3p release experiments were conducted with or without MMP2 enzyme, and 1 mL of
dialysate was taken at different time points (1 hour to 72 hours), followed by 1 mL of
fresh dialysate. A fluorescence microplate reader was used to measure the collected
dialysate at 488/425 nm excitation and emission wavelengths, and the release amount
was calculated. Cumulative release analysis showed that under the action of 300 nM of
MMP2 enzyme, MAIN showed about 75% release within 24 hours, but only about 30%
release in PBS (Figure 2G). The environmental response characteristics of MMP2
enzyme promoted the nanomedicines to release miR-126-3p (nucleotide sequence:
UCGUACCGUGAGUAAUAAUGCG SEQ ID NO. 4) and exerted its effect in specific
tumor microenvironments. SDS-PAGE protein analysis of a series of membrane protein
markers (Fig. 2E) showed that the characteristic proteins inherited from the red blood
cell membranes exhibited good retention in the REMAIN protein spectrum, while the
red blood cell membranes modified nanoparticles REMAIN exhibited membrane protein labeling, endowing the nanoparticles with cell-like functions.
[0046] Embodiment 3: Determination of miR-126-3p expression levels in
nanomedicines MAIN and REMAIN
[0047] NCI-H1299 cells were seeded into 6-well plates at 1x10' cells per well and
cultured in DMEM complete medium. After 24 hours, incubated respectively with PBS,
Lipo3000/miR-126-3p, Main, REMAIN-NC, or free miR-126-3p (miR-126-3p is
equivalent to 150 nM) for 48 hours, and then the expression level of miR-126-3p was
measured by RT-qPCR. The RT-qPCR results showed that the expression level of miR
126-3p was significantly increased in the MAIN and REMAIN groups, and the effect
was equivalent to that of the positive control group Lipo3000 (Fig. 3).
[0048] Embodiment 4: Determination of expression levels of different types of
microRNAs in cells after wrapping with polypeptides and cell membranes
[0049] NCI-H1299 cells were seeded into 6-well plates, at 1 X 105 cells per well, and
cultured in DMEM complete medium. After 24 hours, cells were incubated respectively
with PBS, Lipo3000/microRNAs, 6R-PLGLAG-6R/microRNAs, RBCM/6R
PLGLAG-6R/miRNA-NC, or free microRNAs (microRNAs equivalent to 150 nM) for
48 hours. After 48 hours, the expression levels of microRNAs were measured by RT
qPCR. The RT-qPCR results showed that the expression levels of red blood cell
membrane-wrapped nanoparticles formed by peptide and microRNAs were equivalent
to that of the positive control group Lipo3000 (Table 1)
Table 1 Expression levels of red blood cell membrane-wrapped nanoparticles
formed by peptide and microRNAs
Relative expression level
6R-PLGLAG- RBCM/6R- RBCM/6R PBS Dissociative Lipo3000 6R/ PLGLAG- PLGLAG microRNAs microRNAs 6R/ miRNA-NC miR-486 1±0.12*** 2.17+0.35*** 15.53+1.05 13.71±1.32 15.41±0.78 1.23±0.38*** miR-338 10.15*** 2.22+0.52*** 14.40+0.78 13.09+1.14 14.75+0.81 1.56+0.42*** miR-148b 1+0.15*** 2.66+0.29*** 18.49+0.77 16.77+1.26 18.71+0.66 1.17+0.31 miR-29 1+0.24*** 2.33+0.29*** 17.96+1.28 16.17+0.93 17.10+1.10 0.95+0.33*** miR-96 1+0.18*** 2.70+0.56*** 14.73+1.23 11.54+1.08* 15.54+0.70 1.32+0.26*** miR-137 1+0.09*** 2.36+0.59*** 15.23+1.04 12.86+1.12* 16.20+0.78 1.24+0.33*** All groups were statistically analyzed and compared with RBCM/6R-PLGLAG
6R/microRNAs, * P<0.05, * * P<0.01, and * * * P<0.001.
[0050] Embodiment 5: Anti-tumor effects of nanoparticles in vivo
[0051] Nanoparticles MAIN, REMAIN, and REMAIN-NC were synthesized using
the optimal method in Embodiment 1. Male BALB/c nude mice were implanted with 2
x 106 of NCI-H1299 cells on the right side. After the tumor grew to about 200mm 3 , the
mice were injected through the tail vein every 3 days with 200 L of saline, REMAIN
NC, and free miR-126-3, respectively. The mice treated with REMAIN-NC and free
miR-126-3p were the control group, those treated with gefitinib (20 mg/kg) were the
positive control group, and those treated with REMAIN and MAIN were the
experimental group (20 g of miRNA per mouse). On the 13th day of administration,
tumor tissues were taken, photographed and weighed. From the tumor images (Fig. 4A),
weights (Fig. 4B), and tumor growth curves (Fig. 4C), it could be seen that the mice
treated with REMAIN-NC or free miR-126-3p showed no significant difference
compared with the saline group, while REMAIN, MAIN, and gefitinib all significantly
inhibited tumor growth. Compared with the MAIN group, the REMAIN group showed
better anti-tumor effect, similar to the gefitinib treatment group. The above results
indicated that REMAIN effectively inhibited tumor growth by delivering miR-126-3p
to tumor tissue.
[0052] And the hematoxylin-eosin staining results showed that all treatments did not
cause significant histological changes in the main organs of the mice, and didn't show significant toxicity to the mice (Fig. 5), indicating that the MMP2 enzyme-responsive biomimetic nanoparticles carrier of the present disclosure had good biological safety.
[0053] The above embodiments are merely illustrative of several implementations of
the present disclosure and the descriptions thereof are more specific and detailed but
cannot therefore be construed as limiting the scope of the present disclosure. It should
be noted that those of ordinary skill in the art may make several variations and
modifications without departing from the concept of the present disclosure, and these
variations and modifications fall within the protection scope of the present disclosure.
Therefore, the protection scope of the present disclosure shall be subject to the
appended claims.
[0054] It will be understood that the terms "comprise" and "include" and any of their
derivatives (e.g. comprises, comprising, includes, including) as used in this
specification, and the claims that follow, is to be taken to be inclusive of features to
which the term refers, and is not meant to exclude the presence of any additional
features unless otherwise stated or implied.
Sequence Listing 1 Sequence Listing Information 1-1 File Name IPGD23GZ0637P4-SEQ.xml 1-2 DTD Version V1_3 1-3 Software Name WIPO Sequence 1-4 Software Version 2.3.0 1-5 Production Date 2023-10-10 1-6 Original free text language code 1-7 Non English free text language code 2 General Information 2-1 Current application: IP CN Office 2-2 Current application: Application number 2-3 Current application: Filing date 2-4 Current application: IPGD23GZ0637P4 Applicant file reference 2-5 Earliest priority application: CN IP Office 2-6 Earliest priority application: 2022110148118 Application number 2-7 Earliest priority application: 2022-08-23 Filing date 2-8en Applicant name Guangzhou Medical University 2-8 Applicant name: Name Guangzhou Medical University Latin 2-9en Inventor name 2-9 Inventor name: Name Latin 2-10en Invention title Polypeptide carriers for delivering nucleic acid drugs, nucleic acid drugs for treating tumors, and preparation methods thereof 2-11 Sequence Total Quantity 4
3-1 Sequences 3-1-1 Sequence Number [ID] 1 3-1-2 Molecule Type AA 3-1-3 Length 10 3-1-4 Features source 1..10 Location/Qualifiers mol_type=protein organism=synthetic construct NonEnglishQualifier Value 3-1-5 Residues RRPLGLAGRR 10 3-2 Sequences 3-2-1 Sequence Number [ID] 2 3-2-2 Molecule Type AA 3-2-3 Length 14 3-2-4 Features source 1..14 Location/Qualifiers mol_type=protein organism=synthetic construct NonEnglishQualifier Value 3-2-5 Residues RRRRPLGLAG RRRR 14 3-3 Sequences 3-3-1 Sequence Number [ID] 3 3-3-2 Molecule Type AA 3-3-3 Length 18 3-3-4 Features source 1..18 Location/Qualifiers mol_type=protein organism=synthetic construct NonEnglishQualifier Value 3-3-5 Residues RRRRRRPLGL AGRRRRRR 18 3-4 Sequences 3-4-1 Sequence Number [ID] 4 3-4-2 Molecule Type RNA 3-4-3 Length 22 3-4-4 Features source 1..22 Location/Qualifiers mol_type=other RNA organism=miR-126-3p NonEnglishQualifier Value 3-4-5 Residues tcgtaccgtg agtaataatg cg 22
Claims (19)
1. A polypeptide carrier for delivering nucleic acid drugs, comprising an amino acid
sequence as shown in SEQ ID NO. 3.
2. Use of the polypeptide carrier of claim 1 in the preparation of a drug for delivery of
a MicroRNA drug.
3. A nucleic acid drug for treating tumors, wherein the drug comprises red blood cell
membrane-wrapped nanoparticles formed by the polypeptide carrier of claim 1 and a
tumor-targeting MicroRNA drug.
4. The nucleic acid drug according to claim 3, wherein the tumor is an MMP2 enzyme
responsive tumor.
5. The nucleic acid drug according to claim 4, wherein the tumor is lung cancer.
6. The nucleic acid drug according to claim 3, wherein the MicroRNA drug is a miR
126-3p mimic.
7. The nucleic acid drug according to any one of claims 3 to 6, wherein the mass ratio
of the polypeptide carrier to the tumor-targeting MicroRNA drug is in the range of 20:1
to 40:1.
8. The nucleic acid drug according to claim 7, wherein the mass ratio of the polypeptide
carrier to the tumor-targeting MicroRNA drug is in the range of 28:1 to 32:1.
9. The nucleic acid drug according to any one of claims 3-8, wherein the mass ratio of
the red blood cell membranes to the nanoparticles is in the range of 5:1 to 30:1.
10. The nucleic acid drug according to claim 9, wherein the mass ratio of the red blood
cell membranes to the nanoparticles is in the range of 8:1 to 12:1.
11. A preparation method of the nucleic acid drug for treating tumors according to claim
3, including the following steps:
(1) obtaining red blood cell membranes;
(2) dissolving a polypeptide carrier in PBS to obtain a polypeptide carrier solution;
(3) dissolving a tumor-targeting MicroRNA drug in nuclease-free water to obtain a
MicroRNA drug solution;
(4) mixing the polypeptide carrier solution and the MicroRNA drug solution according
to a mass ratio of the polypeptide carrier to the tumor-targeting MicroRNA drug in the range of 20:1 to 40:1, and letting it stand at room temperature to allow the MicroRNA drug and the polypeptide carrier to form nanoparticles through self-assembly of positive and negative charges;
(5) adding the red blood cell membranes to the obtained nanoparticles, mixing evenly,
and squeezing the prepared nanoparticles through gradient extrusion to obtain
nanoparticles with a uniform particle size to obtain a nucleic acid drug.
12. The preparation method according to claim 11, wherein the mass ratio of the red
blood cell membranes to the nanoparticles is in the range of 5:1 to 30:1.
13. A method of treating a tumor in a subject in need thereof, comprising administering
to said subject a drug comprising red blood cell membrane-wrapped nanoparticles
formed from the polypeptide carrier of claim 1 and a tumor-targeting MicroRNA drug,
wherein the tumor is an MMP2 enzyme-responsive tumor.
14. The method of claim 13, wherein the tumor is lung cancer.
15. The method of claim 13 or 14, wherein the MicroRNA drug is a miR-126-3p mimic.
16. The method of any one of claims 13 to 15, wherein the mass ratio of the polypeptide
carrier to the tumor-targeting MicroRNA drug is in the range of 20:1 to 40:1.
17. The method of claim 16, wherein the mass ratio of the polypeptide carrier to the
tumor-targeting MicroRNA drug is in the range of 28:1 to 32:1.
18. The method of any one of claims 13 to 17, wherein the mass ratio of the red blood
cell membranes to the nanoparticles is in the range of 5:1 to 30:1.
19. The method of claim 18, wherein the mass ratio of the red blood cell membranes to
the nanoparticles is in the range of 8:1 to 12:1.
Fig.1
1/4
Fig.2
Fig.3
2/4
Fig.4
3/4
Fig.5
4/4
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| PCT/CN2023/114224 WO2024041526A1 (en) | 2022-08-23 | 2023-08-22 | Polypeptide vector for delivering nucleic acid medicament, nucleic acid medicament for treating tumor, and preparation method therefor |
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| WO2014145242A1 (en) * | 2013-03-15 | 2014-09-18 | The Board Of Trustees Of The University Of Illinois | Peptide-coated polymer carriers |
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