A dual-responsive nanogel for pre-treatment of exhaled breath condensate samples and a preparation method thereof

Abstract

This invention relates to the field of bioseparation technology, and discloses a dual-responsive nanogel for the pretreatment of exhaled breath condensate samples and its preparation method. The nanogel uses N-isopropylacrylamide as its main framework, and constructs a three-dimensional intelligent network with dual response by introducing pH-responsive monomers and oxidative stress-sensitive crosslinking agents. This material remains in a swollen state under physiological conditions, and only in a pathological microenvironment with pH < 6.8 and H₂O₂ > 100 μM, through the synergistic effect of charge attraction and violent volume contraction, achieves in-situ physical capture and locking of ultra-low abundance markers (such as exosomes) in exhaled breath condensate samples. This invention eliminates the dependence on high-energy-consuming centrifugation and complex microfluidic equipment, possesses high specificity and high enrichment rate, and provides new sample pretreatment conditions for early home screening and rapid bedside auxiliary detection of major respiratory diseases.

Description

Technical Field

[0001] This invention relates to the field of bioseparation technology, specifically to a dual-response nanogel for pretreatment of exhaled breath condensate samples and its preparation method. Background Technology

[0002] Exhaled breath condensate, as a completely non-invasive sample that directly reflects the lung's biochemical microenvironment, is rich in key biomarkers such as proteins, nucleic acids, and extracellular vesicles. It holds significant clinical value and application prospects in the early screening, disease progression monitoring, and environmental exposure assessment of respiratory diseases. However, because exhaled breath condensate is over 99% water, the concentration of target biomarkers is extremely low. Furthermore, it is highly susceptible to non-specific interference from oral impurities, environmental dust, and aerosols during sampling, leading to core technical bottlenecks in its clinical translation due to insufficient sensitivity and low signal-to-noise ratio.

[0003] Existing exhaled breath condensate pretreatment technologies mainly rely on traditional methods such as offline condensation capture, high-speed centrifugation, or lyophilization concentration. These methods are not only cumbersome and heavily reliant on heavy equipment, but also prone to denaturation or loss of unstable biological samples during complex physical processing, failing to meet the real-time requirements of point-of-care testing. Furthermore, most commercially available enrichment materials operate on a passive adsorption model, lacking intelligent identification and dual-response capabilities for endogenous pathological characteristics of samples (such as pH and oxidative stress (ROS) levels), making it difficult to simultaneously achieve precise target analyte locking and automated impurity filtration at the sampling end. Therefore, there is an urgent need in this field to develop an intelligent system that integrates sampling and pretreatment, capable of achieving in-situ enrichment and high-rate concentration of target analytes based on a logic-gated dual-response mechanism, to address the current situation of cumbersome processes and limited sensitivity in exhaled breath condensate detection. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention aims to provide a dual-response nanogel for the pretreatment of exhaled breath condensate samples and its preparation method. This nanogel, by controlling the polymer monomer ratio and crosslinking density, constructs in situ a three-dimensional intelligent molecular sieve network with dual pathological signal response characteristics. This approach abandons the traditional pretreatment mode that relies on high-energy centrifuges, precision pressure pumps, and cumbersome elution steps. Instead, it utilizes the synergistic effect of the sample's endogenous biochemical signals (pH / ROS) and the nanogel's volume phase transition mechanism to achieve automated filtration of non-pathological background components in complex samples (such as exhaled breath condensate) and high-rate, non-destructive in-situ capture of target biomarkers (such as exosomes).

[0005] As one aspect of the present invention, the present invention provides a dual-response nanogel for pretreatment of exhaled breath condensate samples:

[0006] The nanogel of this invention comprises a temperature-sensitive polymer, a pH-sensitive polymer, and an oxidative stress-sensitive crosslinking agent;

[0007] The temperature-sensitive polymer is N-isopropylacrylamide (NIPAM).

[0008] The pH-sensitive polymer is dimethylaminoethyl methacrylate (DMAEMA).

[0009] The oxidative stress-sensitive crosslinking agent is a diacrylate containing borate ester bonds.

[0010] The nanogel is in a hydrophilic swelling state under physiological conditions. Under the dual triggering conditions of pH < 6.8 and H2O2 > 100 μM, the overall structure undergoes a phase transition from hydrophilic to hydrophobic and produces volume shrinkage, thereby achieving physical encapsulation and retention of target markers in exhaled condensate.

[0011] The nanogel exhibits a volume shrinkage rate greater than 50% after being triggered by a dual-response signal.

[0012] In another aspect, the present invention provides a method for preparing a dual-response nanogel for pretreatment of exhaled breath condensate samples, the specific steps of which are as follows:

[0013] (1) Preparation of raw material solution: Dissolve the main monomer NIPAM, pH-responsive monomer DMAEMA and oxidative stress sensitive crosslinking agent diacrylate containing borate ester bond in deionized water, and add an appropriate amount of surfactant to control the particle size;

[0014] (2) Preparation of gel by precipitation polymerization: Under a nitrogen protective atmosphere, the system is heated to 70 °C, and a thermal initiator is added to start the free radical polymerization reaction, so that the monomers are cross-linked in situ in the aqueous phase to form a nanogel emulsion;

[0015] (3) Purification and excipient treatment: After the reaction product is quenched in an ice-water bath, it is subjected to deep dialysis using a dialysis bag with a molecular weight cutoff of 14,000 Da to remove unreacted monomers and impurities; the purified emulsion is then freeze-dried under vacuum to obtain a loose powder product.

[0016] As a preferred embodiment of the preparation method of the dual-response nanogel described in this invention: the mass ratio of NIPAM, DMAEMA and diacrylate containing borate ester bonds in step (1) is 20:(2-4):1.

[0017] As a preferred embodiment of the preparation method of the dual-response nanogel described in this invention: the surfactant in step (1) is sodium dodecyl sulfate.

[0018] As a preferred embodiment of the preparation method of the dual-response nanogel described in this invention: the thermal initiator in step (2) is potassium persulfate or ammonium persulfate.

[0019] As a preferred embodiment of the preparation method of the dual-response nanogel described in this invention, the free radical polymerization reaction in step (2) takes 6 h.

[0020] As another aspect of the present invention, the present invention provides an application of a dual-response nanogel for the pretreatment of exhaled breath condensate samples, the specific steps of which are as follows:

[0021] (1) The nanogel was used as a pretreatment reagent and mixed with the collected exhaled breath condensate sample;

[0022] (2) By utilizing endogenous pathological signals in the sample or by adjusting the sample environment to trigger the contraction of the nanogel, the target marker is captured in situ and locked within the nanogrid;

[0023] (3) Remove non-target impurities through a washing step, and then use an eluent to re-swell the nanogel and release the concentrated target marker.

[0024] The target marker is exosomes.

[0025] The preprocessing process is completed in situ at the sampling end, realizing integrated processing of sampling and marker enrichment.

[0026] Compared with the prior art, the advantages of the present invention are as follows:

[0027] First, this approach achieves highly specific target identification based on a biochemical logic-gated dual-response mechanism. Experimental results show that the nanogel, through its built-in "pH / ROS" dual-response mechanism, can accurately identify the environment in which the sample is located. Compared to traditional materials based on non-specific physical adsorption or single chemical bond capture (which are susceptible to oral impurities and environmental interference), this invention only initiates the capture procedure when a pathological threshold is met (e.g., pH < 6.8 and H2O2 > 100 μM), significantly reducing false positive background interference.

[0028] Second, this method achieves efficient in-situ concentration through an intelligent phase transition mechanism. Relying on the dramatic volume shrinkage of the nanogel from a "hydrophilic swelling state" to a "hydrophobic dense state," this invention achieves physical targeting of target markers (such as exosomes) in dilute exhaled breath condensate samples in a short time. Experimental data clearly show that the local concentration of the target marker is increased after treatment compared to the original sample.

[0029] Third, this scheme achieves the directional capture of the target substance into the nanonetwork through pH-responsive polymers, and synergistically generates a spatial obstruction effect through oxidative stress-induced pore shrinkage, ultimately achieving physical locking. Experiments have confirmed that this synergistic response logic of "induction first, then locking" significantly improves the capture efficiency compared to single-response materials. Attached Figure Description

[0030] Figure 1 This is a particle size response diagram of the nanogel of the present invention.

[0031] Figure 2 This invention improves the capture efficiency of target markers.

[0032] Figure 3 This invention is used to test the specificity of the exhaled breath condensate sample environment.

[0033] Figure 4 This is a statistical chart showing the recovery rate of the target substance in this invention. Detailed Implementation

[0034] The technical solutions described in this invention will now be clearly and completely described with reference to the accompanying drawings of the embodiments of this invention. Obviously, the embodiments described in this specification are only a part of the feasible technical solutions of this invention. Other implementation methods obtained by those skilled in the art based on the embodiments of this invention without any creative effort should be considered to fall within the scope of protection of this invention.

[0035] Example 1: A method for preparing dual-response nanogels for pretreatment of exhaled breath condensate samples

[0036] (1) Preparation of raw material solution: Dissolve 1 g of NIPAM, 0.1 g of pH-responsive monomer DMAEMA, and 0.05 g of diacrylate containing borate ester bonds, a crosslinking agent sensitive to oxidative stress, in 100 mL of deionized water. Then add 0.02 g of sodium dodecyl sulfate surfactant to adjust the micelle concentration. Transfer the mixture to a three-necked flask, purge with nitrogen at 300 rpm for 30 min to remove oxygen, and then heat to 70 °C to build a polymerization environment;

[0037] (2) Preparation of gel by precipitation polymerization: After the system temperature stabilizes, 30 mg of potassium persulfate thermal initiator is added at once to start the polymerization; the reaction is continued for 6 h at 70 ℃ under nitrogen protection. As the linear segments grow and crosslink in situ, the system undergoes a physical phase transition from transparent to milky white, and finally a uniform nanoscale network emulsion is formed.

[0038] (3) Purification and excipient treatment: After the reaction, the mixture was rapidly quenched in an ice-water bath to fix the particle morphology. The product was initially filtered through a 200 nm microporous membrane to remove impurities. Subsequently, the emulsion was placed in a 14000 Da dialysis bag and dialyzed in deionized water for 72 h, with the solution changed every 12 h to remove impurities. The purified product was pre-frozen at -80 ℃ and freeze-dried under vacuum to obtain a loose powder product.

[0039] Example 2: A method for preparing dual-response nanogels for pretreatment of exhaled breath condensate samples

[0040] The difference between this embodiment and Example 1 is that the mass of the pH-responsive monomer DMAEMA is 0.2 g, and the thermal initiator is ammonium persulfate.

[0041] Comparative Example 1: A method for preparing dual-response nanogels for pretreatment of exhaled breath condensate samples

[0042] The difference between this comparative example and Example 1 is that it lacks the pH-responsive monomer DMAEMA described in step (1).

[0043] Comparative Example 2: A method for preparing dual-response nanogels for pretreatment of exhaled breath condensate samples

[0044] The difference between this comparative example and Example 1 is that the diacrylate containing borate ester bonds in the oxidative stress sensitive crosslinking agent in step (1) is replaced with the common crosslinking agent methylenebisacrylamide.

[0045] Comparative Example 3: A method for preparing dual-response nanogels for pretreatment of exhaled breath condensate samples

[0046] The difference between this comparative example and Example 1 is that the diacrylate containing borate ester bonds in the oxidative stress sensitive crosslinking agent in step (1) is replaced with the common crosslinking agent methylenebisacrylamide, and the pH-responsive monomer DMAEMA is missing.

[0047] Comparative Example 4: A method for preparing dual-response nanogels for pretreatment of exhaled breath condensate samples

[0048] The difference between this comparative example and Example 1 is that the pH-responsive monomer DMAEMA in step (1) is replaced with the strong cationic monomer methacryloyloxyethyltrimethylammonium chloride, and the diacrylate containing borate ester bonds of the oxidative stress-sensitive crosslinking agent is missing.

[0049] Experimental Example 1: Particle Size Response Test of Nanogels

[0050] The triggered response capability of the nanogels under specific pathological conditions was evaluated by monitoring the hydrodynamic particle size changes of each group of nanogels under environmental switching. The nanogels obtained in Examples 1-2 and Comparative Examples 1-4 were placed in acetate buffer (pathological environment) at pH 6.5 containing 100 μM H2O2 to measure the initial particle size (D1); the final particle size (D2) was observed after 15 min. The particle size was calculated using the formula V = [1 − (D2 / D1)]. 3 The volume shrinkage rate of each group was calculated by multiplying the result by 100%. The entire experiment was conducted at a constant temperature of 37 ℃, and each group was measured in parallel three times and the average value was taken.

[0051] like Figure 1 As shown, under pathological simulation conditions, Examples 1 and 2 exhibited characteristic "swelling followed by sudden shrinkage" kinetics, with volume shrinkage rates exceeding 90%. This is attributed to the protonation of DMAEMA in acidic conditions triggering initial network relaxation, followed by the breaking of chemical bonds in the oxidative stress-sensitive crosslinking agent, leading to the overall collapse and hydrophilic-hydrophobic transition of the gel network. In contrast, Comparative Example 1, lacking a pH-responsive monomer, showed a significantly slower shrinkage rate, while Comparative Examples 2 and 3, using rigid, ordinary crosslinking agents, exhibited particle size fluctuations of less than 8% throughout the process, demonstrating that the introduction of oxidative stress-sensitive sites is the fundamental driving force for achieving structural physical phase transitions. Although Comparative Example 4 carried a charge, it maintained a stable swollen state due to the lack of a responsive crosslinking network. Therefore, the nanogel prepared in Example 1 can achieve a synergistic response to dual environmental signals.

[0052] Experimental Example 2: Target Marker Acquisition Efficiency Test

[0053] The simulated exhaled breath condensate sample used ultrapure water as a substrate, with sodium chloride, potassium chloride, ammonium sulfate, and a small amount of mucin added to simulate the matrix environment of exhaled breath condensate. A549 cell-derived exosomes labeled with Alexa Fluor 488 fluorescence were added to the simulated sample as target markers. Equal amounts of the nanogels obtained in Examples 1-2 and Comparative Examples 1-4 were added to the sample and incubated for 15 min at 37 ℃, pH 6.5, and with 100 μM H2O2 as a trigger condition. The dual responsiveness of the nanogels was utilized to induce their capture of sparsely distributed exosomes. After incubation, the system was filtered using a 200 nm microporous membrane to retain the shrunken nanogel complex. The filtrate was collected, and the fluorescence intensity (I2) of the residual exosomes in the filtrate was measured using a fluorescence spectrophotometer. Combined with the total fluorescence intensity (I0) of the original sample, the capture efficiency of each material for the target marker was calculated using the formula E = (1 − I2 / I0) × 100%.

[0054] like Figure 2As shown, the nanogels prepared in Examples 1 and 2 achieved capture efficiencies of 93.8% and 95.5%, respectively. This indicates that in the initial stage of the response, the protonation of DMAEMA gives the nanogel a positive charge, rapidly pre-enriching negatively charged exosomes on and inside the network surface through electrostatic attraction. Subsequently, the rapid volume shrinkage induced by oxidative stress tightly encapsulates and locks the exosomes within the dense polymer network. In contrast, although Comparative Example 1 possesses physical shrinkage capabilities, it lacks charge guidance and cannot actively capture exosomes in dilute exhaled condensate samples, resulting in a capture rate as low as 28.5%. While Comparative Example 2, using a common crosslinking agent, exhibits charge attraction, the lack of a physical phase transition process allows exosomes to easily leak out of the loose network due to fluid shear forces during filtration. In summary, the nanogel prepared in Example 1, due to its synergistic dual-response properties, offers certain advantages in the pretreatment of complex, low-abundance clinical samples.

[0055] Experiment Example 3: Test of Environmental Specificity Recognition Ability

[0056] To verify the recognition ability of the nanogel prepared in Example 1 in non-pathological environments, the nanogels obtained in Examples 1-2 and Comparative Examples 1-4 were placed in simulated exhaled breath condensate samples from healthy individuals. This sample environment simulated a normal physiological environment (pH 7.4 and no H2O2). Equal amounts of A549 cell-derived exosome markers labeled with Alexa Fluor 488 were added to each group of samples. The fluorescence intensity of the added fluorescently labeled exosome markers was defined as Itotal. The samples were incubated at 37 °C in the dark for 15 min, simulating the screening process of clinical sampling. Subsequently, each group of materials was retained using a 200 nm microporous membrane. The residual fluorescence in the filtrate was detected using a fluorescence spectrophotometer, and the residual fluorescence intensity was defined as Irest. The non-specific adsorption rate of each group of materials was calculated using the formula D = [1 − (Irest / Itotal)] × 100% to evaluate the recognition ability of the materials in normal non-pathological environments.

[0057] like Figure 3 As shown, under physiological conditions at pH 7.4, the nonspecific adsorption rates of Examples 1 and 2 were both below 10%, indicating that the nanogels prepared in Examples 1-2 possess excellent environmental recognition capabilities and can effectively avoid false positive interference. Although the adsorption rates of Comparative Examples 1-3 were low (<4%), combined with Experiment 2, it is clear that they also lacked capture capabilities under pathological conditions. However, the nonspecific adsorption rate of Comparative Example 4 was as high as 79.5%, indicating that traditional charged materials lack environmental discrimination capabilities and will exhibit forced adsorption even in healthy samples, leading to a serious risk of false positive diagnoses. In summary, the nanogel prepared in Example 1 does not initiate capture behavior under normal physiological conditions, avoiding the risk of false positive diagnoses and significantly improving the accuracy of clinical auxiliary diagnosis.

[0058] Experimental Example 4: Elution and Recovery Test of Target Biomarkers

[0059] To evaluate the practical performance of the nanogel prepared in Example 1 in converting low-abundance biomarkers into high-concentration samples, nanogel complexes of A549 cell-derived exosomes captured in Experiment 2 (Examples 1, Comparative Examples 2 and 3) were collected. The complexes were first washed three times with deionized water at pH 7.4 to remove nonspecific adsorbates. Subsequently, the complexes were redispersed in 200 μL of elution buffer (pH 8.0 buffer containing 0.1 M fructose) and shaken at 37 °C in the dark for 15 min. Fructose molecules competitively bind to cross-linking sites in the gel network, inducing the nanogel to re-swell from a contracted state, thereby releasing the encapsulated exosomes. The supernatant was collected by centrifugation, and the recovered exosome content (Mr) was detected using a fluorescence spectrophotometer. Combined with the initial total mass of the target biomarker in the original sample (M0), the recovery rate was calculated using the formula R (recovery rate) = (Mr / M0) × 100%.

[0060] like Figure 4 As shown, the biomarker recovery rate of Example 1 reached over 88.6%, successfully achieving "noise reduction" and concentration of ultra-low abundance biomarkers. In contrast, Comparative Examples 2 and 3, lacking an effective "physical locking" mechanism, suffered biomarker loss due to fluid shear during the washing step, resulting in a final recovery rate of less than 15%. Therefore, this demonstrates that the nanogel prepared in Example 1 can transform trace signals in complex backgrounds into high-concentration, easily detectable, high-quality samples.

[0061] In summary, this invention constructs a nanogel with dual-response properties and utilizes its volume shrinkage effect triggered by specific pathological signals (pH / ROS) to achieve in-situ specific capture and efficient enrichment of extremely low abundance biomarkers in exhaled breath condensate. This approach not only eliminates the dependence on complex external power equipment in traditional pretreatment, significantly improving the portability and integration of sample processing, but also solves the pain points of poor specificity, high background noise, and easy degradation of biomarkers in clinical auxiliary testing through controllable charge attraction and physical locking mechanisms, providing a brand-new materials science platform for highly sensitive non-invasive biochemical auxiliary diagnosis.

[0062] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the principle of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. A dual-response nanogel for pretreatment of exhaled breath condensate samples, characterized in that, Nanogels include temperature-sensitive polymers, pH-sensitive polymers, and oxidative stress-sensitive crosslinking agents; The temperature-sensitive polymer is N-isopropylacrylamide; The pH-sensitive polymer is dimethylaminoethyl methacrylate; The oxidative stress-sensitive crosslinking agent is a diacrylate containing boron ester bonds; The nanogel is in a hydrophilic swelling state under physiological conditions. Under the dual triggering conditions of pH < 6.8 and H2O2 > 100 μM, the overall structure undergoes a phase transition from hydrophilic to hydrophobic and produces volume shrinkage, thereby achieving physical encapsulation and retention of target markers in exhaled condensate.

2. The dual-response nanogel as described in claim 1, characterized in that, The volume shrinkage rate of the nanogel after dual-response signal-triggered shrinkage is greater than 50%.

3. A method for preparing a dual-response nanogel for pretreatment of exhaled breath condensate samples, characterized in that, The preparation method includes the following steps: (1) Preparation of raw material solution: The main monomer N-isopropylacrylamide, pH-responsive monomer dimethylaminoethyl methacrylate and oxidative stress sensitive crosslinking agent diacrylate containing borate ester bond are dissolved in deionized water, and surfactants are added to control the particle size. (2) Preparation of gel by precipitation polymerization: Under a nitrogen protective atmosphere, the system is heated to 70 °C, and a thermal initiator is added to start the free radical polymerization reaction. After 6 h, the monomer is cross-linked in the aqueous phase to form a nanogel emulsion. (3) Purification and excipient treatment: After the reaction product is quenched in an ice-water bath, it is subjected to deep dialysis using a dialysis bag with a molecular weight cutoff of 14,000 Da to remove unreacted monomers and impurities; the purified emulsion is then freeze-dried under vacuum to obtain a loose powder product.

4. The method for preparing the dual-response nanogel as described in claim 3, characterized in that, The mass ratio of N-isopropylacrylamide, dimethylaminoethyl methacrylate, and diacrylate containing boron ester bonds in step (1) is 20:(2-4):

1.

5. The method for preparing the dual-responsive nanogel as described in claim 3, characterized in that, The surfactant in step (1) is sodium dodecyl sulfate.

6. The method for preparing the dual-responsive nanogel as described in claim 3, characterized in that, The thermal initiator in step (2) is potassium persulfate or ammonium persulfate.

7. An application of the dual-response nanogel as described in any one of claims 1-2 in the pretreatment of exhaled breath condensate samples, comprising the following specific steps: (1) The nanogel was used as a pretreatment reagent and mixed with the collected exhaled breath condensate sample; (2) By utilizing endogenous pathological signals in the sample or by adjusting the sample environment to trigger the contraction of the nanogel, the target marker is captured in situ and locked within the nanogrid; (3) Remove non-target impurities through a washing step, and then use an eluent to re-swell the nanogel and release the concentrated target marker.

8. The application according to claim 8, characterized in that, The target marker is exosomes.

9. The application according to claim 8, characterized in that, The preprocessing process is completed in situ at the sampling end, realizing integrated processing of sampling and marker enrichment.

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