Hemoglobin-based micro-nano reactor as well as preparation method and application thereof

Abstract

The invention discloses a hemoglobin-based micro-nano reactor as well as a preparation method and application thereof, belongs to the technical field of biochemistry, and particularly relates to a hemoglobin-based micro-nano reactor which is formed by self-assembly of hemoglobin-polylactic acid conjugates. The hemoglobin-based micro-nano reactor has a cell-like space structure and has multiple functions of a drug delivery system and a micro-nano reactor, hemoglobin biomolecules in the hemoglobin-based micro-reactor are utilized to catalyze atom transfer radical polymerization reaction, and the activity of the hemoglobin-based micro-nano reactor is improved under the condition of not additionally adding a catalyst or gene modification. The micro-nano reactor has the advantages of simplicity and convenience in synthesis, good biocompatibility, various catalytic polymers and the like, and is suitable for the fields of cell engineering, biological material preparation, fluorescence labeling, production of polymers by a fermentation method and the like.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry technology, and in particular relates to a hemoglobin-based micro / nano reactor, its preparation method, and its application. Background Technology

[0002] Intracellular polymerization, as a bioorthogonal technique, allows for the direct synthesis of polymers within living cells, providing a novel approach for cell function modification, the development of new biomaterials, and synthetic biology research. Traditional intracellular polymerization methods often rely on photoinitiation systems or exogenous inorganic catalysts, which suffer from harsh reaction conditions, poor biocompatibility, and potential cell damage. Furthermore, existing methods frequently require the expression of specific enzymes through gene modification as catalysts, making the process complex and costly.

[0003] Hemoglobin is the most abundant natural protein in living organisms, possessing advantages such as wide availability, ease of extraction, and functional tunability. Hemoglobin has a unique iron-containing structure, exhibiting potential catalytic activity. Utilizing hemoglobin's natural catalytic ability to trigger polymerization reactions, without the need for gene modification or exogenous catalysts, would significantly simplify the intracellular polymerization process and improve biocompatibility. How to efficiently deliver hemoglobin, monomers used in polymer synthesis, and initiators into cells to construct a stable polymerization system within the cell and achieve efficient and controllable polymerization reactions is a pressing technical challenge in this field.

[0004] Micro and nanocarriers have broad application prospects in drug delivery, biocatalysis, tissue engineering, and other fields. Among them, protein-based nanocarriers have become a research hotspot in recent years due to their good biocompatibility, biodegradability, and functional tunability. Therefore, in this field, it is hoped to develop a hemoglobin-based micro and nanocarrier that can efficiently carry monomers and initiators used in polymer synthesis, and deliver them into cells to act as a micro / nanoreactor for efficient and controllable intracellular polymerization reactions. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a hemoglobin-based micro / nano reactor, its preparation method, and its applications. This hemoglobin-based micro / nano reactor possesses a cell-like spatial structure, combining the multiple functions of a drug delivery system and a micro / nano reactor. Utilizing the hemoglobin biomolecules within the microreactor to catalyze atom transfer radical polymerization reaction, polymers can be synthesized within living cells without the need for additional catalysts or genetic modification. This micro / nano reactor offers advantages such as simple synthesis, good biocompatibility, and the ability to catalyze a variety of polymers, making it suitable for applications in cell engineering, biomaterial preparation, fluorescent labeling, and fermentation-based polymer production.

[0006] To achieve the above objectives, the present invention provides a hemoglobin-based micro / nano reactor, which is formed by the self-assembly of hemoglobin-polylactic acid conjugate.

[0007] Preferably, the particle size of the hemoglobin-based micro / nano reactor is 170 nm to 2 μm, and the hemoglobin-based micro / nano reactor has a hollow vesicle structure.

[0008] Preferably, the hemoglobin-polylactic acid conjugate is prepared by covalently linking hemoglobin subunits with alkenyl-terminated polylactic acid.

[0009] More preferably, the molecular weight of the alkenyl-terminated polylactic acid is 15,000 or 28,000.

[0010] This invention also provides a method for preparing the hemoglobin-based micro / nano reactor, comprising the following steps: 1) preparing alkenyl-terminated polylactic acid by atom transfer radical polymerization; 2) covalently linking hemoglobin with the alkenyl-terminated polylactic acid obtained in step 1) to form a hemoglobin-polylactic acid conjugate; 3) dispersing the hemoglobin-polylactic acid conjugate obtained in step 2) in a solvent to obtain system A; 4) dispersing the polymerization active monomer and polymerization initiator in water to obtain system B; 5) adding system A obtained in step 3) dropwise to system B obtained in step 4) to obtain system C; 6) dialyzing system C obtained in step 5) to obtain the hemoglobin-based micro / nano reactor.

[0011] Preferably, the polymerizable active monomer in step 4) includes one or more of acrylamide, N-isopropylacrylamide, N-acryloylmorpholine, hydroxyethyl methacrylate, and hydroxypropyl acrylate.

[0012] Preferably, the polymerization initiator in step 4) includes one or more of α-bromophenylacetic acid ethyl ester, 2-hydroxyethyl-2-bromoisobutyrate, α-bromophenylacetic acid, and brominated polyethylene glycol.

[0013] The present invention also provides the application of the hemoglobin-based micro / nano reactor in catalytic atom transfer radical polymerization.

[0014] Preferably, the critical aggregation concentration of the hemoglobin-based micro / nano reactor is 0.018~0.024 mg / mL.

[0015] Preferably, the reaction system is a hemoglobin-based micro / nano reactor, which catalyzes atom transfer radical polymerization at a controllable temperature, terminates the reaction by introducing air, and extracts the polymer using an organic solvent.

[0016] Compared with the prior art, the present invention has the following advantages and technical effects: This invention provides a hemoglobin-based micro / nanoreactor, its preparation method, and its applications. The hemoglobin-based micro / nanoreactor is constructed by self-assembling a conjugate (Hb-PLA) formed by covalent bonds between hemoglobin (Hb) and polylactic acid (PLA) into a hollow vesicle structure similar to a cellular bilayer. It can encapsulate polymerization monomers, and its surface can be modified with targeting molecules. It possesses pH-responsive dissociation characteristics, drug-targeted delivery capabilities, and nanoreactor catalytic activity. With a cell-like spatial structure, it combines the multiple functions of a drug delivery system and a micro / nanoreactor, utilizing the hemoglobin biomolecules within the hemoglobin-based microreactor to catalyze atom transfer radical polymerization (ATRP). This invention relates to cell engineering, biomaterial preparation, fluorescent labeling, and fermentation-based polymer production. The hemoglobin-based micro / nano reactor can biosynthesize temperature-responsive polymers (such as PNIPAm) and biocompatible polymers (such as PHPMA) for biomaterial preparation; it can also functionally modify cells through intracellular polymerization, such as fluorescent labeling and intracellular polymer network construction, for synthetic biology research; and it can catalytically synthesize polymers using the hemoglobin-based micro / nano reactor, providing new preparation routes for bio-based materials and drug delivery carriers. Utilizing natural hemoglobin molecules as a catalyst eliminates the need for exogenous enzymes or inorganic catalysts, simplifying the process and reducing costs. The reaction conditions are mild, with polymerization occurring at physiological temperatures (37°C) and under anaerobic conditions, consistent with the cellular physiological environment. Good biocompatibility is achieved through optimized initiator and monomer concentrations, ensuring high cell viability (≥90%). The polymerization process does not affect cell survival and proliferation, exhibiting excellent bioorthogonality. Polymerization is highly controllable; by selecting different initiator-monomer combinations, various types of polymers can be synthesized, with controllable product molecular weight and dispersion (dispersion as low as 1.51). Monomer conversion can be adjusted by regulating reaction time (up to 90%). Visual monitoring and multifunctional expansion are possible through the introduction of fluorescent monomer copolymerization systems, allowing direct monitoring of the polymerization reaction and intracellular polymeric localization via fluorescence signals. The synthesized fluorescent polymers can be used for long-term cell tracking, facilitating cell engineering applications. With a wide range of applications, it can be used in the biosynthesis of functional polymers, cell modification, and biomaterial development, providing a new technological platform for the cross-integration of synthetic biology and polymer chemistry. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1This is a schematic diagram illustrating the preparation method of hemoglobin-based micro / nano reactors by self-assembly of hemoglobin-polylactic acid conjugates according to the present invention; Figure 2 This is the 1H NMR spectrum of the alkenyl-terminated polylactic acid synthesized in this invention; Figure 3 This is the GPC diagram of the alkenyl-terminated polylactic acid synthesized in this invention; Figure 4 These are transmission electron microscope (TEM) images of the hemoglobin-based micro / nano reactor of the present invention. In the images, A is a TEM image of the conjugate Hb-PLA15000 with a scale bar of 500 nm, B is a particle size distribution of the conjugate Hb-PLA15000, C is a TEM image of the conjugate Hb-PLA28000 with a scale bar of 2 μm, and D is a particle size distribution of the conjugate Hb-PLA28000. Figure 5 This invention uses a pyrene fluorescent probe method to determine the critical aggregation concentration of Hb-PLA15000 and Hb-PLA28000 dispersions, where A represents the excitation spectrum of pyrene in Hb-PLA15000 solutions of different concentrations, and B represents the fluorescence intensity of pyrene. 334 / I 332 A graph was plotted against the concentration of Hb-PLA15000, where C represents the excitation spectrum of pyrene in Hb-PLA28000 solutions of different concentrations, and D represents the fluorescence intensity I of pyrene. 334 / I 332 Plot the concentration of Hb-PLA28000; Figure 6 This invention uses nuclear magnetic resonance spectroscopy to detect the conversion efficiency of N-isopropylacrylamide polymerization catalyzed by ATRP in a hemoglobin-based micro / nano reactor in solution; Figure 7 In this invention, GPC was used to determine the average molecular weight of the polymerized product PEG-PNIPAM in solution; Figure 8 This invention uses nuclear magnetic resonance spectroscopy to detect the conversion efficiency of hemoglobin-based micro / nanoreactors in cells via ATRP-catalyzed polymerization of N-isopropylacrylamide; Figure 9 In this invention, GPC was used to detect the average molecular weight of the polymer PEG-PNIPAM in cells; Figure 10 To demonstrate the fluorescence intensity of hemoglobin-based micro / nano reactors in cells after polymerization of fluorescein O-methacrylate and N-isopropylacrylamide via ATRP catalysis, the present invention uses flow cytometry to detect the fluorescence intensity of the reactors. In the figure, Control represents group B, and P (NIPAm-co-FOM) represents group A. Figure 11The present invention uses a hemoglobin-based micro / nano reactor to catalyze the polymerization of fluorescent monomers fluorescein O-methacrylate and N-isopropylacrylamide in cells via the ATRP reaction. After cell lysis, the copolymer is purified by size exclusion chromatography to obtain the characteristic broad-spectrum fluorescence of P(NIPAm-co-FOM). In the figure, Control represents group B and P(NIPAm-co-FOM) represents group A. Figure 12 In this invention, GPC was used to detect the average molecular weight of the polymer P (NIPAm-co-FOM) in cells. Detailed Implementation

[0019] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, for numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range. Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art described herein. While only preferred methods and materials are described herein, any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of reference to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0020] The PBS buffer used in this invention was purchased from Beyotime Biotechnology, and its formulation was: 135 mM NaCl, 4.7 mM KCl, 10 mM Na₂HPO₄, 2 mM NaH₂PO₄, pH 7.3 ± 0.1. The DMF solution used in this invention was purchased from Maclean's, 99% biotechnology grade. The RPMI-1640 cell culture medium used in this invention was purchased from Thermo Fisher Scientific's Gibco series. The 4T1 tumor cells used in this invention were purchased from Wuhan Pronosun Biotechnology Co., Ltd.

[0021] Example 1 Polymerization of hemoglobin-based micro / nanoreactors in solution: Preparation of alkenyl-terminated polylactic acid (Alkenyl-PLA): 5g of lactide was added, along with 0.1g of Sn(Oct)₂ as a catalyst and 0.2g of alkenyl-termining agent. The reaction was carried out at 110℃ for 24h to obtain Alkenyl-PLA. The 1H NMR spectrum is shown in the figure (e.g., [image of Alkenyl-PLA]). Figure 2The successful synthesis was verified. GPC characterization showed that the number-average molecular weight (NMR) of polylactic acid (PLA) was 7000, and the PDI was 1.64; the NMR of PLA was 10000, and the PDI was 1.67; the NMR of PLA was 15000, and the PDI was 1.51; and the NMR of PLA was 28000, and the PDI was 1.59 (e.g., ...). Figure 3 ).

[0022] Synthesis of hemoglobin-polylactic acid conjugate (Hb-PLA): 10 mg of hemoglobin (Hb) was weighed and dissolved in 10 mL of PBS buffer at pH 7.4 to obtain a hemoglobin solution. Alkenyl-PLA was dissolved in 0.5 mL of DMF solution to obtain an Alkenyl-PLA solution. The Alkenyl-PLA solution was added dropwise to the hemoglobin solution (the molar ratio of Hb to PLA was 1:1). The mixture was stirred at room temperature for 12 h and dialyzed through a dialysis bag (molecular weight cutoff of 10 kDa) for 24 h to obtain Hb-PLA.

[0023] Preparation of hemoglobin-based micro / nano reactors (e.g.) Figure 1 Hb-PLA15000, a conjugate of PLA with a number-average molecular weight of 15000, was dispersed in PBS buffer at a concentration of 1 mM and incubated at 37°C for 4 h. Nanoparticle hollow vesicles were observed by TEM and characterized by DLS. The vesicle particle size was 170–220 nm (e.g., ...). Figure 4 China A and Figure 4 (B) The conjugate Hb-PLA28000, with a number-average molecular weight of 28000, was dispersed in PBS buffer at a concentration of 1 mM and incubated at 37°C for 4 h. Micron-sized hollow vesicles were observed by TEM, and DLS characterization showed that the vesicle particle size was 1.5-2 μm (e.g., ...). Figure 4 C and Figure 4 (D). Using the pyrene fluorescent probe method, pyrene was dissolved in a dispersion of PLA conjugates Hb-PLA15000 and Hb-PLA28000, with a number-average molecular weight of 15000, at a concentration of 6 × 10⁻⁶. - 7 mol / L, the fluorescence spectra of pyrene at different concentrations were measured, with I 334 / I 332 Plotting the ratio against concentration yields a critical aggregation concentration (CAC) of 0.0224 mg / mL for Hb-PLA15000. Figure 5 China A and Figure 5 (From B), the critical aggregation concentration (CAC) of Hb-PLA28000 was found to be 0.018 mg / mL (e.g., ...). Figure 5 C and Figure 5 (D).

[0024] Construction of the reaction system for the hemoglobin-based micro / nanoreactor in solution: N-isopropylacrylamide monomer and brominated polyethylene glycol (PEG200-Br) initiator were dissolved in water at a monomer concentration of 50 mM and an initiator concentration of 1 mM. Hb-PLA15000 was dispersed in the monomer / initiator solution at a Hb concentration of 1 mM.

[0025] ATRP catalysis in solution using a hemoglobin-based micro / nanoreactor: The reaction system was placed in a 37°C water bath and stirred for 0 h, 3 h, 6 h, 12 h, 24 h, and 36 h. Air was introduced into the reaction solution at different time points to terminate the reaction; the air quenched residual free radicals and terminated chain growth. The solution was extracted with dichloromethane, repeated five times. The organic solvent phase obtained from the extraction was evaporated to remove dichloromethane, and the polymerization product was purified and collected using size exclusion chromatography. Samples were then taken for further analysis. 1 H NMR and GPC characterization. 1 ¹H NMR showed that after 36 h of reaction, the vinyl peak at 5.6–6.2 ppm disappeared, while the isopropyl peak at 3.8–3.9 ppm increased, indicating a monomer conversion rate of 91% (e.g., ...). Figure 6 GPC showed that the polymer PEG-PNIPAM had an Mn of 12900 and a PDI of 1.51 (e.g., ...). Figure 7 ).

[0026] Example 2 Polymerization of hemoglobin-based micro / nanoreactors in cells: The synthesis of Hb-PLA and the preparation of hemoglobin-based micro / nano reactors were the same as in Example 1.

[0027] Construction of the hemoglobin-based micro / nanoreactor reaction system in cells: N-isopropylacrylamide monomer and brominated polyethylene glycol (PEG200-Br) initiator were dissolved in water at a concentration of 50 mM and 1 mM, respectively. Hb-PLA15000 was dispersed in the monomer / initiator solution at a concentration of 1 mM. The hemoglobin-based micro / nanoreactor was extracted by ultracentrifugation (20000 r / min) and then dispersed in RPMI-1640 cell culture medium at a concentration of 50 mM monomer, 1 mM initiator, and 1 mM Hb. 2 × 10⁻⁶ 6 Four 4T1 tumor cells were seeded into 10cm cell culture dishes and cultured in RPMI-1640 cell culture medium at 37°C for 24 hours. The culture medium was removed from the 6-well plate, and then 10mL of culture medium containing hemoglobin-based micro / nanoreactors was added to the cell culture dish for co-culture with the 4T1 cells.

[0028] ATRP catalysis in cells using hemoglobin-based micro / nanoreactors: Co-cultured 4T1 cells were placed in a 37°C incubator with a gas composition of 5% CO2, 2.5% O2, and 92.5% N2. Cells were cultured for 0, 3, 6, 12, 24, and 36 hours. At different time points, cells were removed, and air was introduced to terminate the reaction. Air quenches residual free radicals and terminates chain growth. Cells were digested, centrifuged at 1000 rpm, and washed with PBS buffer to remove extracellular initiators and monomers. Cells were then lysed with dichloromethane, and the polymers were extracted from the cells, repeated five times. The extracted organic solvent phase was evaporated to remove dichloromethane, and the polymerization product was purified and collected using size exclusion chromatography. Samples were then taken for further processing. 1 HNMR and GPC characterization. 1 ¹H NMR showed that after 36 h of reaction, the vinyl peak at 5.6–6.2 ppm disappeared, while the isopropyl peak at 3.8–3.9 ppm increased, indicating a monomer conversion rate of 75% (e.g., ...). Figure 8 GPC showed that the polymer PEG-PNIPAM had an Mn of 9700 and a PDI of 1.82 (e.g., ...). Figure 9 ).

[0029] Example 3 Fluorescent copolymerization reaction of hemoglobin-based micro / nanoreactors in cells: The synthesis of Hb-PLA and the preparation of hemoglobin-based micro / nano reactors were the same as in Example 1.

[0030] Construction of the reaction system for hemoglobin-based micro / nanoreactors in cells: Two groups were established. Group A: Fluorescent monomer O-methacrylate, monomer N-isopropylacrylamide, and initiator brominated polyethylene glycol (PEG200-Br) were dissolved in water. The concentrations of the fluorescent monomers were 5 mM and 45 mM, respectively, and the initiator concentration was 1 mM. Hb-PLA15000 was dispersed in the monomer / initiator solution, with an Hb concentration of 1 mM. Group B: Fluorescent monomer O-methacrylate and monomer N-isopropylacrylamide were dissolved in water. The concentrations of the fluorescent monomers were 5 mM and 45 mM, respectively. The Hb-PLA15000 conjugate was dispersed in the monomer / initiator solution, with an Hb concentration of 1 mM.

[0031] Hemoglobin-based micro / nanoreactors were extracted by ultracentrifugation (20000 r / min). The extracted hemoglobin-based micro / nanoreactors were dispersed in RPMI-1640 cell culture medium with a fluorescent monomer concentration of 5 mM, a monomer concentration of 45 mM, an initiator concentration of 1 mM, and a Hb concentration of 1 mM. 2 × 10⁻⁶ 6Four 4T1 tumor cells were seeded into 10cm cell culture dishes and cultured in RPMI-1640 cell culture medium at 37°C for 24 hours. The culture medium was removed from the 6-well plate, and then 10mL of culture medium containing hemoglobin-based micro / nanoreactors was added to the cell culture dish for co-culture with the 4T1 cells.

[0032] The reaction was terminated by purging with air, and 4T1 cells were collected by centrifugation at 1000 r / min. After washing with PBS, the cells were analyzed by flow cytometry. The results showed that the fluorescence intensity of the polymerized cells (P(NIPAm-co-FOM)) was 9.7 times that of the control group (without initiator), indicating that the fluorescent copolymer was successfully synthesized and localized intracellularly (e.g., ...). Figure 10 ).

[0033] After cell lysis, the copolymer was purified by size exclusion chromatography. The resulting P(NIPAm-co-FOM) exhibited characteristic fluorescence emission at 520 nm under 488 nm excitation (e.g., Figure 11 GPC analysis showed that its Mn=9100 and PDI=1.64 (e.g. Figure 12 ).

[0034] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A hemoglobin-based micro / nano reactor, characterized in that, The hemoglobin-based micro / nano reactor is formed by the self-assembly of hemoglobin-polylactic acid conjugate.

2. The hemoglobin-based micro / nano reactor according to claim 1, characterized in that, The hemoglobin-based micro / nano reactor has a particle size of 170 nm to 2 μm and is a hollow vesicle structure.

3. The hemoglobin-based micro / nano reactor according to claim 1, characterized in that, The hemoglobin-polylactic acid conjugate is prepared by covalently linking hemoglobin subunits with alkenyl-terminated polylactic acid.

4. The hemoglobin-based micro / nano reactor according to claim 3, characterized in that, The molecular weight of the alkenyl-terminated polylactic acid is 15,000 or 28,000.

5. The method for preparing the hemoglobin-based micro / nano reactor according to any one of claims 1 to 4, characterized in that, Includes the following steps: 1) Preparation of alkenyl-terminated polylactic acid via atom transfer radical polymerization; 2) Hemoglobin and the alkenyl-terminated polylactic acid obtained in step 1) are covalently linked to form a hemoglobin-polylactic acid conjugate; 3) Disperse the hemoglobin-polylactic acid conjugate obtained in step 2) in a solvent to obtain system A; 4) Disperse the polymerization active monomer and polymerization initiator in water to obtain system B; 5) Add system A obtained in step 3) dropwise to system B obtained in step 4) to obtain system C; 6) Dialyze the system C obtained in step 5) to obtain a hemoglobin-based micro / nano reactor.

6. The preparation method according to claim 5, characterized in that, The polymerizable active monomers mentioned in step 4) include one or more of acrylamide, N-isopropylacrylamide, N-acryloylmorpholine, hydroxyethyl methacrylate, and hydroxypropyl acrylate.

7. The preparation method according to claim 5, characterized in that, The polymerization initiator mentioned in step 4) includes one or more of α-bromophenylacetic acid ethyl ester, 2-hydroxyethyl-2-bromoisobutyrate, α-bromophenylacetic acid, and brominated polyethylene glycol.

8. The application of the hemoglobin-based micro / nano reactor as described in any one of claims 1 to 4 in catalytic atom transfer radical polymerization.

9. The application according to claim 8, characterized in that, The critical aggregation concentration of hemoglobin-based micro / nano reactors is 0.018~0.024 mg / mL.

10. The application according to claim 8, characterized in that, Specifically, the reaction system is a hemoglobin-based micro / nano reactor. The reaction is catalyzed by atom transfer radical polymerization at a controlled temperature. The reaction is terminated by introducing air, and the polymer is extracted using an organic solvent.

Images