Preparation of functionalized mesoporous silica nanoparticles for mass tag applications in mass cytometry

Abstract

The present disclosure provides functionalized mesoporous silica nanoparticles (MSNs) and methods of making functionalized MSNs. MSNs are post-synthetically modified with a zwitterionic silane and a polyethylene glycol (PEG) silane to inhibit non-specific protein adsorption. The PEG silane can be a long-chain PEG 5k silane, and the zwitterionic silane can be a zwitterionic sulfobetaine silane. The MSNs can be loaded with at least one lanthanide ion for use as a mass tag reagent for mass cytometry. The MSNs can be conjugated to antibodies; for example, the MSNs can comprise azido-PEG 5k silane conjugated to dibenzocyclooctyne-functionalized anti-biotin Abs (DBCO-anti-biotin Abs).

Description

Background Technology

[0001] In modern biomedical research, understanding the complexities of cell biology, disease mechanisms, and immune responses has become more important than ever. To address these challenges, scientists have been dedicated to developing innovative technologies to identify cellular biomarkers, thereby providing deeper insights into the diverse and complex world of biology. Among these groundbreaking technologies, mass cytometry (MC) has emerged as a powerful tool with the potential to revolutionize single-cell analysis.

[0002] MC is a cutting-edge bioanalytical technique that combines the principles of flow cytometry with atomic mass spectrometry, enabling researchers to simultaneously measure multiple parameters at the single-cell level. In this method, cells are first stained with heavy-metal isotope-labeled antibodies (Abs). Subsequently, the single-cell suspension is introduced into an inductively coupled plasma time-of-flight mass spectrometer (ICP-TOF-MS), where multiple target biomarkers can be examined by monitoring signals in different and discrete mass channels. By replacing traditional fluorophores with heavy metal isotopes as antibody tags, MC overcomes several inherent limitations of flow cytometry, such as spectral overlap interference and limited multiplex detection capabilities. In principle, MC is capable of simultaneously detecting more than 100 parameters in a single experiment, depending on the number of various stable heavy metal isotopes with masses ranging from m / z 75 to 209. However, due to the lack of carriers capable of capturing heavy metal isotopes, currently only about 50 parameters can be identified on a conventional basis. Therefore, there has long been an urgent need to develop novel elemental mass labeling reagents to expand detection channels and enhance characterization and multiplexing capabilities. Summary of the Invention

[0003] In various embodiments, the MSN-based nanoparticles disclosed herein provide highly sensitive mass-tagged reagents. Advantages of the disclosed MSN-based nanoparticles include (but are not limited to): low degree of nonspecific binding (NSB) and successful attachment of antibodies to MSNs. In various embodiments, antibodies are associated with PMSNs-Zwi-PEG. 5K The coupling of -N3 is advantageous for use as a biomarker in mass flow cytometry.

[0004] In various embodiments and examples, compositions comprising functionalized mesoporous silica nanoparticles (MSNs) are provided, wherein the functionalized MSN comprises: mesoporous silica nanoparticles (MSNs); at least one zwitterionic silane; and at least one long-chain polyethylene glycol (PEG) functionalized silane. In various embodiments, the at least one zwitterionic silane is a silane containing a zwitterionic betaine group. In various embodiments, the at least one zwitterionic betaine group-containing silane is a zwitterionic carboxybetaine silane. In various embodiments, the at least one zwitterionic betaine group-containing silane is a zwitterionic sulfobetaine silane. In various embodiments, the long-chain polyethylene glycol (PEG) functionalized silane is a PEG. 5k Silane, and preferably long-chain PEG silane. In various embodiments, the PEG... 5k Silane is azide-PEG 5k -Silane.

[0005] In various embodiments, zwitterionic silanes have the following characteristics: (a) one or more quaternary ammonium groups (-N) + (CH3)3) or other cationic moieties; (b) one or more sulfonic acid groups (-SO3) - ), carboxylic acid group (-COO) - (c) or phosphate groups; (d) medium chain length (e.g., C3-C6), for example to balance hydrophilicity and structural integrity; and (e.g., one or more hydrolyzable groups, such as trimethoxysilane or triethoxysilane.

[0006] As used herein, the term "long-chain" in the context of PEG refers to PEG with a molecular weight greater than about 1.5 thousand Daltons (kDa), greater than about 3 kDa, greater than about 4 kDa, and in various embodiments, the molecular weight is in the range of about 3 kDa to about 10 kDa.

[0007] In various forms and embodiments, the functionalized MSN further comprises lanthanide ions, such as those selected from the group consisting of La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, and combinations thereof. In various embodiments, each of the functionalized MSNs contains at least about 1 × 10⁻⁶ ions. 4 Up to at least approximately 7.4 × 10 4 One lanthanide ion.

[0008] In various forms and embodiments, functionalized MSNs as disclosed herein are provided conjugated with antibodies (Abs). In various embodiments, compositions are provided in which the functionalized MSN is conjugated with dibenzocyclooctyne-functionalized anti-biotin Abs (DBCO-anti-biotin Abs), and in various embodiments, the functionalized MSN comprises azide-PEG conjugated with the dibenzocyclooctyne-functionalized anti-biotin Abs. 5K -Silane.

[0009] In various forms and embodiments, the multifunctional mesoporous silica nanoparticles (MSNs) disclosed herein are produced using zwitterionic silanes and (in various embodiments) long-chain PEG. 5K The silane is post-modified. In various embodiments, the functionalized MSN is prepared by post-modification with zwitterionic sulfobetaine silane.

[0010] To enhance their colloidal stability in buffer solutions and reduce nonspecific binding (NSB) to cells, a series of surface modifications of PMSNs disclosed in this paper provide compositions suitable for mass cytometry applications. As-synthesized PMSNs-Zwi-mPEG with a diameter of 48 nm are also described. 5K The nanoparticles have been lyophilized and redispersed in H2O and 1×PBS buffer solution with good stability. In various embodiments, these functionalized MSNs exhibited excellent capacity to carry large amounts of lanthanide ions (e.g., Tb, 19.6 mg / g) with negligible ion loss in H2O (approximately 1.4%) and 1×PBS buffer (approximately 0.2%). Furthermore, pretreatment of Tb@PMSNs-Zwi-mPEG with PBS buffer... 5K After 20 minutes, the nanoparticles significantly reduced the leaching of Tb ions into various other buffer solutions (e.g., HEPES buffer, MES buffer, Bis-tris buffer) to less than 1%, presumably due to the precipitation of TbPO4. These nanoparticles have been found to be versatile in loading various lanthanide elements, thus providing novel elemental mass tags in various embodiments.

[0011] Among the various forms, functionalized MSN adopts PEG. 5K Chain. In various embodiments, PMSNs-Zwi-mPEG5K PEG with 0.52 chains / nm² 5K Chain density showed the lowest degree of human serum albumin (HSA) adsorption (1.1 wt%), indicating (unbound by theory) that this dense brush conformation can effectively inhibit protein adsorption.

[0012] In various states of samples, azido-PEG was used. 5K -Silane was used to modify mPEG 5K -Silane substitution to prepare PMSNs-Zwi-PEG 5K -N3 nanoparticles. These nanoparticles were then reacted with DBCO-modified antibiotin antibodies to generate unique NP-Ab conjugates. All of these conjugates retained their antibody function and were able to bind to the target biotin Cy5 molecule. In various embodiments, the bioconjugation reaction was carried out at 25°C for 4 hours, where a 50:1 Ab to NP ratio was observed to provide high conjugation efficiency while retaining a large degree of antibody function.

[0013] In various embodiments, the resulting methoxy-terminated nanoparticles (PMSN-Zwi-mPEG) 5K This demonstrates that each nanoparticle (NP) can carry up to 7.4 × 10⁻⁶. 4 The ability to generate Tb ions was negligible, with ion loss observed in both H2O and 1×PBS buffer. In various embodiments, the self-loading of Tb by PMSN-Zwi-mPEG in various buffers was suppressed by first immersing the nanoparticles in 1×PBS buffer. 5K Detected exudation. In various embodiments, mPEG is provided on the surface of nanoparticles. 5K The composition and chain density provide a high grafting density and reduce nonspecific binding to serum proteins. In various embodiments, azido-PEG attachment is provided. 5K -Silane nanoparticles (PMSN-Zwi-PEG) 5K -N3), which can be conjugated to dibenzocyclooctyne-functionalized anti-biotin antibodies (DBCO-anti-biotinAbs) via click chemistry. In various embodiments, the purified NP-Ab conjugates can efficiently bind biotin Cy5 molecules, as can be demonstrated, for example, by a distinct peak corresponding to Cy5 under UV-vis detection.

[0014] In various forms and embodiments, methods for analyzing cells are also provided, the methods comprising introducing cells into a composition comprising a functionalized MSN as disclosed herein.

[0015] In various embodiments, compositions comprising functionalized MSNs used in methods for analyzing cells comprise mixtures of functionalized MSNs, said mixture comprising a plurality of functionalized MSNs, each functionalized MSN being an embodiment of a functionalized MSN as disclosed herein. In various embodiments, such mixtures comprise at least 10, 100, 1,000, 10,000, 100,000, 1,000,000, or 10,000,000 functionalized MSNs. In various embodiments, the functionalized MSNs in said mixture of multiple functionalized MSNs comprise: mesoporous silica nanoparticles (MSN); at least one zwitterionic silane; and at least one long-chain polyethylene glycol (PEG) functionalized silane. In various embodiments, said at least one zwitterionic silane is a zwitterionic sulfobetaine silane. In various embodiments, said long-chain polyethylene glycol (PEG) functionalized silane is PEG. 5K -Silane. In various embodiments, the PEG 5K -Silane is azide-PEG 5K -Silane. In various embodiments, the functionalized MSN comprises zwitterionic sulfobetaine silane and long-chain PEG. 5K Silane.

[0016] In various forms and embodiments, at least a portion of the functionalized MSNs in the plurality of functionalized MSN mixtures further comprise lanthanide ions, such as lanthanide ions selected from the group consisting of La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, and combinations thereof. In various embodiments, the lanthanide ion is Tb, and each such functionalized MSN has at least about 1 x 10⁻⁶ lanthanide ions. 4 Up to at least approximately 7.4 x 10 4 One Tb ion.

[0017] In various forms and embodiments, at least a portion of the functionalized MSNs in the plurality of functionalized MSN mixtures are conjugated with antibodies (Abs). In various embodiments, such functionalized MSNs are conjugated with dibenzocyclooctylene-functionalized anti-biotin antibodies (DBCO-anti-biotinAbs), and in various embodiments, such functionalized MSNs comprise azide-PEG conjugated with dibenzocyclooctylene-functionalized anti-biotin antibodies. 5K -Silane. Attached Figure Description

[0018] The invention will be described in conjunction with the following drawings, wherein similar reference numerals denote similar elements, and wherein: Figure 1 A schematic diagram of a program for creating a multi-functional MSN.

[0019] Figure 2 It has multiple subgraphs, among which Figure 2 A and Figure 2 B is the TEM image; Figure 2 C and Figure 2 D is the size distribution histogram; Figure 2 E and Figure 2 F represents the redispersed PMSNs nanoparticles (dh=73.0 nm, PDI=0.14) and the redispersed PMSNs-Zwi-mPEG, respectively. 5K DLS results for nanoparticles (dh=81.9nm, PDI=0.11).

[0020] Figure 3 It has multiple subgraphs, where Figure 3 Example A illustrates the room-temperature synthesis of zwitterionic sulfobetaine silane (SBS); Figure 3 B and Figure 3 C represents the 1H NMR and ¹³C NMR spectra of the original SBS synthesized in Examples 1-13, respectively.

[0021] Figure 4 It has multiple sub-images, which are TEM images, among which Figure 4 A displays PMSNs, Figure 4 B is higher than Figure 2 Magnification of A and 2B shows PMSNs-Zwi-mPEG 5K .

[0022] Figure 5 It has multiple subgraphs, among which Figure 5 A is a digital photograph of the dried powder and the redispersed PMSNs colloidal solution (20 mg / mL); Figure 5 B shows the hydrodynamic diameter distribution of the synthesized original PMSNs solution (dh=70.5 nm, PDI=0.097) and the redispersed PMSNs solution (dh=73.0 nm, PDI=0.14); Figure 5 C shows the redispersed PMSNs-Zwi-mPEG 5K Stability of H2O (hollow square symbol) and 1×PBS buffer (hollow circle symbol) stored at 4°C for 7 days; Figure 5 D shows the repeatability test of PMSNs nanoparticle synthesis. Figure 5 The error bars in D represent one standard deviation of the particle size distribution.

[0023] Figure 6 It has multiple subgraphs, among which Figure 6A shows PMSNs nanoparticles, PMSNs-Zwi nanoparticles, and PMSNs-Zwi-mPEG before and after CTAC removal. 5K FTIR spectra of nanoparticles; Figure 6 B displays bare MSNs, PMSNs, PMSNs-Zwi, and PMSNs-Zwi-mPEG. 5K TGA curves of nanoparticles; Figure 6 C shows the nitrogen adsorption-desorption isotherm; Figure 6 D shows the PMSNs, PMSNs-Zwi, and PMSNs-Zwi-mPEG calculated by the Barrett-Joyner-Halenda (BJH) method. 5K Pore ​​size distribution of nanoparticles.

[0024] Figure 7 The FTIR spectra of PMSNs in Examples 1-11 are shown before removal of the CTAC template and after two extraction cycles in a mixed solution of concentrated hydrochloric acid and ethanol.

[0025] Figure 8 The 1H NMR spectra of various nanoparticle suspensions are shown (acquired on a 600MHz Agilent DD2 NMR spectrometer); among them Figure 8 From bottom to top, the contents are: PMSN nanoparticles in D2O (20 mg / mL), PMSN-Zwi nanoparticles in D2O (5 mg / mL), and PMSNs-Zwi-mPEG in D2O. 5K Nanoparticles (5 mg / mL).

[0026] Figure 9 Display of various lanthanides in PMSNs-Zwi-mPEG 5K Loading amount on nanoparticles. A mixture of lanthanides at a concentration of 1 mg / mL was mixed with nanoparticles (0.5 mg / mL) for 24 hours. The error bar represents a standard deviation, calculated from three independent samples, with each sample subjected to three repeated measurements.

[0027] Figure 10 It has multiple subgraphs, among which Figure 10 A shows PMSNs-Zwi-mPEG without PBS buffer pretreatment. 5K Tb of nanoparticles in different aqueous environments and under various conditions 3+Ion effusion distribution: 0.1M Na2CO3 / NaHCO3 buffer (pH 10.6), 0.1M MES buffer (pH 5.5), 0.02M Bis-Tris buffer (pH 6.5), 0.01M MEPES buffer (pH 7.4), H2O and 1×PBS; Figure 10 B shows the PMSNs-Zwi-mPEG without pretreatment (solid line) and with pretreatment (dashed line). 5K Tb of nanoparticles in 0.1M Na2CO3 / NaHCO3 buffer (pH 10.6) 3+ Comparison of exudation distribution maps; Figure 10 C displays PMSNs-Zwi-mPEG 5K TEM image of nanoparticles immersed in 0.1M Na2CO3 / NaHCO3 buffer (pH 10.6) 4 days ago; Figure 10 D displays PMSNs-Zwi-mPEG 5K TEM image of nanoparticles after immersion in 0.1M Na2CO3 / NaHCO3 buffer (pH 10.6) for 4 days.

[0028] Figure 11 It has multiple subgraphs, among which Figure 11 A shows the PMSNs-Zwi-mPEG after different pretreatment times (i.e., 20 minutes, 40 minutes, 60 minutes, and 120 minutes). 5K Nanoparticles exudate Tb into 0.1M MES buffer (pH 5.5). 3+ The case of ions; Figure 11 B shows that PMSNs-Zwi-mPEG was pretreated in 1×PBS for 20 minutes. 5K Tb of nanoparticles in different aqueous environments and under various conditions 3+ Ion effusion distribution maps: 0.1M Na2CO3 / NaHCO3 buffer (pH 10.6), 0.1M MES buffer (pH 5.5), 0.02M Bis-tris buffer (pH 6.5), 0.01M HEPES buffer (pH 7.4). The distribution maps for H2O and 1×PBS buffer are untreated.

[0029] Figure 12 It has multiple subgraphs, among which Figure 12 A shows the composition of different mPEGs. 5K PMSNs-Zwi-mPEG synthesized by dosage 5K The TGA curve. Figure 12 The number following the sample name in A indicates the nanoparticles and mPEG. 5K The mass ratio, for example, 1010 indicates NP to mPEG.5K The mass ratio is 10:10; Figure 12 B shows from Figure 12 The TGA data shown in Figure A is exported from the grafted mPEG. 5K Quantitative analysis of content was performed, expressed as weight percentage (wt%) and grafting density (chain number / nm²). Figure 12 C shows bare MSNs, PMSNs-Zwi, and PMSNs-Zwi-mPEG with different compositions. 5K Quantitative analysis of the adsorbed HSA. Figure 12 In C, the error bars represent a standard deviation calculated from three independent samples, each sample being measured three times repeatedly; and Figure 12 Schematic diagram of the PEG conformation on the surface of nanoparticles: PMSNs-Zwi-mPEG 5K Brush-like (left) and dense brush-like conformations (right).

[0030] Figure 13 It has multiple subgraphs, among which Figure 13 A shows the calibration curve for the Pierce™ BCA protein assay, used to quantify the amount of protein adsorbed by nanoparticles; Figure 13 B shows the calibration curve for the MicroBCA™ protein assay used to quantify the number of antibiotin antibodies / control antibodies labeled on nanoparticles.

[0031] Figure 14 This shows that antibiotin antibody and PMSNs-Zwi-PEG can be combined. 5K A schematic diagram of a procedure for coupling -N3 nanoparticles with Cy5 biotin molecules for antigen recognition testing.

[0032] Figure 15 It has multiple subgraphs, among which Figure 15 A represents the preparation of PMSNs-Zwi-mPEG. 5K PMSNs-Zwi-PEG prepared under substantially similar reaction conditions 5K TEM image of -N3 nanoparticles; Figure 15 B shows PMSNs-Zwi-PEG 5K - Size distribution histogram of N3 nanoparticles (d =49.1±3.1nm, CV=6.2%). Figure 15 C shows PMSNs-Zwi-PEG redispersed in PBS buffer. 5K -N3 nanoparticles (dh=79.9nm; PDI=0.095) and PMSNs-Zwi-PEG stored in PBS buffer for 7 days 5KDLS results for -N3 nanoparticles (dh=79.8nm; PDI=0.81); Figure 15 D shows PMSNs-Zwi-PEG in 1×PBS buffer. 5K UV-vis spectrum of Cy5 nanoparticles.

[0033] Figure 16 It has multiple subgraphs, among which Figure 16 A represents the UV-vis spectra (above A280) of the DBCO-modified anti-biotin antibody and the DBCO-modified isotype control antibody. Each anti-biotin antibody and control antibody has approximately four DBCO groups. Figure 16 B shows the DLS results of the sample in 1×PBS buffer: anti-biotin antibody (dh=10.7nm, PDI=0.036), PMSNs-Zwi-PEG. 5K -N3 nanoparticles (dh=79.9nm, PDI=0.095), NP-antibiotin antibody conjugate (room temperature (RT), 4h, 22:1, dh=89.6nm, PDI=0.13) and NP-antibiotin antibody conjugate (RT, 4h, 50:1, dh=90.6nm, PDI=0.12); Figure 16 C shows the UV-vis spectra of NP-antibiotin antibody-Cy5, NP-control antibody-Cy5, and the corresponding NP-antibiotin antibody conjugate without Cy5 biotin (RT, 4h, 50:1). Figure 16 The dashed curve in C represents the modeling baseline for NP-antibiotin antibody-Cy5, obtained by least-squares regression of the corresponding NP-antibiotin antibody spectrum, followed by normalization to match the antibiotin antibody-Cy5 trace. Inset: Magnified (500 to 800 nm); Figure 16 D shows the number of Cy5 on each antibody after bioconjugation and purification under various conditions with Ab to NP feed ratios of 22:1 and 50:1. Figure 16 The error bars in D represent a standard deviation determined from repeated measurements of three independent samples. Detailed Implementation

[0034] Current elemental labeling strategies for designing mass-labeled reagents are primarily based on metal-chelating polymers (MCPs) and various types of nanoparticles (NPs). In the case of MCPs, only a limited number of metals, including Ln (La to Lu, except Pm), Y, In, Bi, Cd, Pt, Re, and Te, can be stably chelated onto the polymer side chains and exhibit good performance in mass cytometry. Despite significant efforts by researchers to incorporate a wider range of metal types into MCPs, major challenges remain, particularly regarding polymer chain cross-linking and potential antibody-antigen recognition disruption. More importantly, the average number of metal atoms per polymer is typically between 20 and 25, resulting in 100–250 metal ions per antibody (Ab). This only allows for efficient detection of cells expressing up to 10-1 metal ions. 4 Up to 10 7 The range of biomarkers limits the lower limit of MC measurement. This is necessary for detecting low-abundance biomarkers (<10 per cell). 4 (e.g., cytokine receptors) require a "brighter" quality tag that can carry more metal atoms.

[0035] This disclosure provides mesoporous silica nanoparticles (MSNs) as a basis for mass cytometry mass labeling. MSNs are silica materials with pores ranging from 2 to 50 nm in diameter. In various embodiments, the synthesis modifies MSNs to a diameter of 45 nm, employing short-chain polyethylene glycol (PEG6-9 silane, PMSN) to promote colloidal stability during preparation, followed by sequential addition of zwitterionic sulfobetaine molecules and long-chain PEG. 5K Polymer-modified PMSNs (PMSN-Zwi-mPEG) 5K This can, for example, reduce nonspecific serum protein adsorption and promote antibody attachment. In various embodiments, Tb ions are loaded into PMSN-Zwi-mPEG in aqueous solution. 5K In the nanoparticles, ion loss observed in 1×PBS buffer was negligible. Furthermore, this paper also reveals the presence of PMSN-Zwi-mPEG. 5k mPEG of NPs 5K Examples of various compositions and chain densities, which can be used, for example, in various embodiments and applications, to minimize the non-specific binding of nanoparticles to human serum albumin (HSA). In various embodiments, N3-PEG is used. 5K Silane-substituted mPEG 5K Silanes to obtain N3-terminated nanoparticles (PMSN-Zwi-PEG) 5K-N3). In various embodiments, the obtained PMSN-Zwi-PEG is processed by click chemistry. 5K -N3 nanoparticles were conjugated to DBCO-modified antibiotin antibodies, and various bioconjugation conditions were provided in various embodiments; for example, this was confirmed in various embodiments by recognition with biotinylated Cy5 molecules. Table 1 summarizes the various PMSN-Zwi-PEGs prepared and discussed herein. 5K Physicochemical properties of nanoparticles.

[0036] Table 1. Series PMSN-Zwi-mPEG 5K Physicochemical characterization of nanoparticles.

[0037]

[0038] Preparation and characterization of multifunctional MSN nanoparticles.

[0039] To function effectively as MC reporters, MSN-based mass tagging reagents need to meet several criteria. One such criterion is the balance between nanoparticle size and the amount of lanthanides loaded. If the nanoparticle size is too small, it may not be able to carry enough lanthanides; while if the nanoparticle size is too large, it may cause an excess of lanthanides beyond the load capacity of the MC instrument. Furthermore, surface modification is required to impart colloidal stability to the nanoparticles in phosphate-buffered saline, reduce non-specific interactions with cells, and facilitate the introduction of appropriate functional groups for antibody conjugation. In the various forms and examples provided herein, MSNs were synthesized via a hydrothermal method using triethanolamine (TEA) as a base catalyst, hexadecyltrimethylammonium chloride (CTAC) as a structure-directing agent, and tetraethoxysilane (TEOS) as a silica precursor. The reaction was carried out at 95 °C, and a short hydrophilic PEG6-9 silane (M=459-591) was introduced via co-condensation to improve the colloidal stability and dispersibility of silica nanoparticles, thereby providing small size (d =45nm), uniform (CV<8%) and redispersible PEG6-9 modified MSN; see Table 1 for example.

[0040] Next, 3-(dimethyl(3-(trimethoxysilyl)propyl)ammonium)propane-1-sulfonate inner salt (a zwitterionic sulfobetaine-silane (SBS))) and a longer-chain polyethylene glycol silane (PEG) were used. 5K These nanoparticles (M=5000) were sequentially treated to enhance colloidal stability and reduce protein adsorption in subsequent applications of these materials. The group of reactions is summarized in... Figure 1 Details are provided in the examples, where Figure 1 Note: In various embodiments, the nanoparticles are co-condensed with PEG. 6-9 Silane is treated to form PMSNs, followed by removal of CTAC. The PMSNs are then treated with SBS to form PMSNs-Zwi, and then those with attached methoxy-PEG... 5K Silane becomes PMSNs-Zwi-mPEG 5K PMSNs-Zwi particles are contacted with lanthanides (Ln) (Tb in this example) to provide Tb@PMSNs-Zwi-mPEG. 5K .

[0041] PMSNs and PMSNs-Zwi-mPEG 5K Transmission electron microscopy (TEM) images and their size distribution histograms are as follows: Figure 2 As shown in A-2D, these PMSNs exhibit a uniform and discrete spherical morphology with an average diameter of 44.5 ± 3.4 nm (CV = 7.7%). Figure 4 In the TEM image shown in Figure A, worm-like mesopore structures can be seen at higher magnification. In this example, SBS and mPEG are used. 5K After surface modification, the nanoparticles maintained their spherical shape and uniform size, with an average diameter of 47.9 ± 3.1 nm (CV = 6.5%). Furthermore, due to the limited contrast of the surface-bound polymer, the outer edges and worm-like mesoporous structure became blurred after the introduction of the long-chain PEG polymer (see [link to article]). Figure 4 B).

[0042] In various embodiments, hydrophilic polymers are introduced to provide lyophilization of the samples and redispersal of the nanoparticles. PMSNs and PMSNs-Zwi-mPEG redispersed in H2O are examples. 5KIt is an optically transparent colloidal solution, and the hydrodynamic dimensions of the PMSNs after drying and redispersing are almost identical to those of the synthesized in-situ PMSNs solution (see, for example...). Figure 5 B). Figure 2 E and 2F show the dynamic light scattering (DLS) results of the redispersed sample in this example. They show a relatively narrow single-peak size distribution, with the PMSNs having a Z-mean diameter dh = 73.0 nm (PDI = 0.14) in H₂O, and PMSNs-Zwi-mPEG. 5K The dh value was 81.9 nm (PDI = 0.11). The dh values ​​of both samples in PBS buffer matched their dh values ​​in H2O very well. As expected, the dh value measured by DLS was greater than the d value. This value is due to the contribution of the hydration layer in aqueous environments. (Compared to PEG) 5K Corona-related hydration layer thickness (L) h It can be estimated as:

[0043] Neither DLS nor TEM results showed any signs of particle aggregation, indicating good stability in an aqueous environment. Furthermore, no precipitation was observed, and the hydrodynamic dimensions of these nanoparticles did not change significantly during a week of storage in PBS buffer. Figure 5 C). To evaluate the reproducibility of PMSNs synthesis, six batches of nanoparticles were prepared under identical conditions, and their size was analyzed by TEM. Figure 5 As shown in D, the average diameter of all six nanoparticle samples (experiment numbers) is approximately 45 nm.

[0044] Fourier transform infrared spectroscopy (FT-IR) was used to confirm the removal of the CTAC template and to characterize subsequent use with SBS and mPEG. 5K Surface finishing work performed. For example... Figure 6 As shown in A and 7, before CTAC extraction, two peaks at 2924 cm⁻¹ attributed to CH stretching vibrations can be observed. - ¹ and 2855cm - ¹. After the first extraction cycle (12 hours in acidic ethanol solution), these peaks decreased significantly, indicating successful removal of the CTAC template. However, even after two extraction cycles, two peaks associated with the -CH2 group remained ( Figure 7 This can be attributed to the PEG introduced into PMSNs during co-condensation. 6-9The -CH2 group in silanes. In various embodiments, SBS and mPEG... 5K Successful functionalization on PMSNs surfaces via 1486 cm - A quaternary ammonium group (-N) appears at position ¹. + (CH3)2R - ) and at 2924cm - ¹ and 2855cm - ¹ appears at position PEG 5K The large peak related to the -CH2 stretching of the chain was verified (see [link]). Figure 6 A). In various embodiments, the silica framework of the multifunctional MSN-based nanoparticles remained substantially unchanged after polymer coating, a fact attributed to the retention of 449 cm⁻¹. - ¹(Si-O bending), 796cm - ¹(Si-O-Si symmetric stretching) and 1055cm - The peak at ¹(Si-O-Si asymmetric stretching) indicates this. Surface modifications were also characterized by ¹H NMR, as shown in... Figure 8 As shown and discussed in examples.

[0045] Thermogravimetric analysis (TGA) was used to quantify the surface modification degree of PMSNs (see [reference]). Figure 6 B, Note Figure 6 The percentages shown for each trace in B refer to the percentage of mass retained. Unmodified bare MSNs were used as the baseline. In the temperature range of 100 to 800 °C, bare MSNs lost 7.4% of their mass (i.e., retained 92.6% of their mass). This mass loss is thought to be primarily due to the removal of adsorbed water and the dehydroxylation of silanol groups. PMSNs showed a more significant weight loss of 23.2%, which was attributed to low molecular weight PEG. 6-9 Degradation of silanes. PMSNs-Zwi and PMSNs-Zwi-mPEG 5K The samples exhibited greater weight losses, at 28.5% and 41.1%, respectively, indicating that PMSNs-Zwi-mPEG... 5K The NPs contained approximately 5.3 wt% SBS and 12.6 wt% mPEG. 5K Unbound by theory, this analysis shows that in various embodiments, zwitterionic sulfobetaine forms only volatile products upon pyrolysis, and indicates that SBS and mPEG are not present in this disclosure. 5K Silane has been successfully immobilized onto the surface of PMSNs nanoparticles.

[0046] In this example, PMSNs, PMSNs-Zwi, and PMSNs-Zwi-mPEG are used. 5K The nitrogen adsorption / desorption isotherms of the sample are shown in Figure 6 In section C, the results are listed in Table 2. All adsorption / desorption plots are typical Type IV isotherms, indicating a well-defined mesoporous structure. A sharp adsorption step at relative pressures (P / P0) between 0.2 and 0.4 is characteristic of mesoporous materials with relatively small pore sizes. A secondary adsorption step at higher P / P0 > 0.8, without theoretical constraints, is attributed in this paper to the gaps formed by the nanoparticles after freeze-drying, termed textural porosity. Furthermore, the capillary condensation behavior within the mesopores of this example sample was determined to be reversible, resulting in a hysteresis loop in the isotherm. It was also determined that after surface modification, the adsorption step became less steep compared to PMSNs, and the inflection point shifted to a slightly lower P / P0, indicating a slight reduction in pore diameter. Figure 6 The pore size distribution curves in D and Table 2 are shown. Therefore, in various embodiments, SBS and mPEG are used. 5K Surface modification of PMSNs with silanes can be used to adjust surface area, pore volume and pore diameter, resulting in gradual reductions from 637, 514 to 229 m² / g, from 0.96, 0.66 to 0.43 cm³ / g, and from 2.7, 2.4 to 2.2 nm, as shown in Table 2, for example.

[0047] Table 2. Summary of surface area, pore volume, and pore size of various NP samples

[0048] The specific surface area is calculated using the BET method based on data within the range of P / P0 < 0.3 (Brunauer, S.; Emmett, PH; Teller, E., “Adsorption of Gases in Multimolecular Layers”, Journal of the American Chemical Society 1938, 60(2), 309-319).

[0049] b. The total pore volume was calculated using the BJH method at P / P0=0.99 (Barrett, EP; Joyner, LG; Halenda, PP, "The Determination of Pore Volume and Area Distributions in Porous Substances I Computations from Nitrogen Isotherms" Journal of the American Chemical Society 1951, 73(1), 373-380).

[0050] The pore size c is specified by the maximum value of the BJH pore size distribution.

[0051] In various embodiments, by treating PMSNs with SBS, the overall pore volume and mean pore diameter are reduced. Without being bound by theory, this is thought to be due to SBS modifying both the inner surface of the pores and the outer surface of the nanoparticles. PEG is determined in various embodiments. 5K The radius of gyration is comparable to the aperture, which is considered to indicate (unbound by theory) that these molecules are end-grafted onto the outer surface of the nanoparticles.

[0052] Assuming these molecules are grafted to the outer surface of the nanoparticles, for PEG on the outer surface of PMSNs 5K The grafting density of the chains is estimated. This calculation uses the PEG of each PMSN. 5K The mass (or quantity) of the polymer is estimated, along with the proportion of the PMSNs' outer surface not occupied by the pores. This estimation assumes the nanoparticles are perfectly spherical and the mesopores are straight cylinders that penetrate the volume. This analysis yields the following equation:

[0053] Where σ PEG / nm² This represents the mPEG per nm² of PMSNs surface area. 5K The grafting density of the chain, ρ represents the density of silica (1.85-2.20 g / cm³), and β is the mPEG in the sample obtained through TGA. 5K The weight ratio (in mg / g), NAV is Avogadro's constant, d (in nm) is the average diameter measured by TEM, and M is mPEG. 5KThe molecular weight is 5000 g / mol, and Vp is the pore volume (0.96 cm³ / g) obtained by nitrogen adsorption / desorption analysis using the BJH method. (Number 10) - ² 4 Convert the units from m² to nm². A more detailed derivation of the equation can be found in Example 12. Since the density ρ of silicon dioxide can range from 1.85 to 2.20 g / cm³... 3 The values ​​vary between 0.52 and 0.84 chains / nm², therefore mPEG is used. 5K Silane estimated value. This PEG 5K Grafting density levels create polymer brushes on the particle surface.

[0054] lanthanides (Ln) in PEGylated MSN 3+ )load.

[0055] In order to make PMSNs-Zwi-mPEG 5K The conversion of nanoparticles into useful reporters for mass cytometry requires the use of Ln. 3+ The ions are labeled. In various embodiments, we add metal ions as follows. We add them from TbCl3·6H2O (10 mg / mL Tb 3+ Tb has only one naturally occurring stable isotope (with 100% abundance). 159 Tb). This facilitates detection via inductively coupled plasma mass spectrometry (ICP-MS).

[0056] PMSNs-Zwi-mPEG as determined by ICP-MS 5K Tb 3+ The maximum loading was approximately 19.6 mg / g, equivalent to 0.123 mmol / g. The number of particles in the solution was determined using a ZetaView Nanoparticle Tracking Analyzer (NTA). The Tb content was compared with the 1.0 × 10¹¹ Tb / g obtained by NTA. 5 The combined concentration of individual nanoparticles results in a maximum Tb loading of approximately 7.4 × 10⁻⁶. 4 Tb 3+ / NP. From a mass cytometry perspective, this is a useful value.

[0057] However, as an effective quality label for mass cytometry, many other criteria must also be met. These include the ability to load different metal isotopes, stability to prevent ion loss during storage or application, surface functionalization for antibody attachment, and surface modifications to minimize or prevent nonspecific binding (NSB) to cells, which are provided in various embodiments herein.

[0058] To test the various PMSNs-Zwi-mPEGs disclosed in this article 5K The nanoparticles exhibit versatility by loading various types of lanthanide ions. A mixture of metal salts containing 1 mg / mL each of LaCl3·7H2O, CeCl3·7H2O, PrCl3·6H2O, NdCl3·6H2O, SmCl3·6H2O, EuCl3·6H2O, GdCl3·6H2O, TbCl3·6H2O, HoCl3·6H2O, Er(NO3)3·5H2O, TmCl3·6H2O, Yb(NO3)3·5H2O, and LuCl3·6H2O was mixed with nanoparticles (0.5 mg / mL) and stirred at room temperature for 24 hours. Note that a large excess of Ln was added. 3+ Ions. The loading content of each lanthanide element in the nanoparticles was measured by ICP-MS, and ICP-MS analysis of the supernatant after separation of the nanoparticles showed that only about 0.1% Ln was present. 3+ Ions are absorbed by nanoparticles. Figure 9 Data in the document shows that, in various embodiments, the PMSNs-Zwi-mPEG provided herein... 5K Nanoparticles can combine the entire lanthanide series of Ln 3+ The ions exhibit some selectivity for heavier lanthanides, particularly Yb. 3+ and Lu 3+ Some of the options.

[0059] Lanthanide ion leaching experiment.

[0060] MSNs designed for drug delivery are engineered to act as drug carriers, but also to release the loaded molecules at the target site. These design goals contradict the needs of MC (Mixed Drug Delivery) applications. For MC applications, MSNs must prevent ion loss during storage and / or application. The stability of various embodiments of metal-loaded MSNs in buffer solution was tested using Tb. Tb ion loss from Tb@PMSNs-Zwi-mPEG was monitored by ICP-MS. 5KLeakage in various buffer solutions within a defined pH range. The buffer range includes 0.1M Na₂CO₃ / NaHCO₃ (pH 10.6), 0.1M MES buffer (pH 5.5), 0.02M Bis-tris buffer (pH 6.5), 0.01M HEPES buffer (pH 7.4), and H₂O to 1×PBS buffer (pH 7.4). Tb 3+ Independent charts of the time distribution of releases are collected in Figure 10 In samples A and 10B, no detectable ion loss was observed in H2O (approximately 1.4%) and 1×PBS buffer (approximately 0.2%) over a 4-day period. However, in 0.01M HEPES buffer at pH 7.4, Tb ions slowly leached from the nanoparticles, reaching approximately 13% after 4 days. For samples in 0.02M MES buffer at pH 6.5 and 0.1M MES buffer at pH 5.5, the Tb ion loss was more significant, reaching 45% and 46%, respectively. Furthermore, when Tb@PMSNs-Zwi-mPEG was present... 5K When nanoparticles were dispersed in 0.1M Na2CO3 / NaHCO3 at pH 10.6, nearly 100% of Tb ions were lost.

[0061] Several methods have been explored to precipitate Tb within the pores to reduce leaching, but several difficulties have been encountered. For example, preparing colloidal hydrogels that confine europium (Eu) ions to the core results in the formation of "insoluble" EuF3 nanoparticles. However, these microgels still retain Eu... 3+ Ions diffuse into the continuous medium. When using Tb... 3+ In the experiment, TbF3 (Ksp(TbF3) is approximately 10) was used. - ¹ 9 It is converted into TbPO4, which has a much lower solubility (Ksp(TbPO4) is approximately 10). - ² 7 The methods described above; however, these attempts to precipitate TbPO4 inside the hydrogel by exposing it to several inorganic phosphate sources resulted in precipitation on the outside of the hydrogel. However, in the present embodiments, it was found that the TbF3-loaded hydrogel is stable in PBS buffer, and dispersing the microgel in PBS buffer provides it with anti-percolation stability and leads to the formation of TbPO4 nanocrystals inside. Figure 10 A shows Tb@PMSNs-Zwi-mPEG 5K Nanoparticles are stable against efflux in PB buffer. (Reference) Figure 10B, also examined Tb@PMSNs-Zwi-mPEG pretreated with PBS. 5K The nanoparticles exhibited anti-leaching stability, and pretreatment was found to substantially reduce Tb leaching loss.

[0062] refer to Figure 11 A, Tb@PMSNs-Zwi-mPEG 5K Nanoparticle samples were suspended in 1×PBS buffer and aged for various times (20 min, 40 min, 60 min, and 120 min), then washed three times with H2O by sedimentation-redispersion (45,000×g, 45 min). The samples were then transferred to 0.1 M MES buffer (pH 5.5) to investigate exudation. Figure 11 As shown in Figure A, regardless of the pretreatment time, the leakage of Tb ions was effectively suppressed to less than 1%. Surprisingly, longer pretreatment times in 1×PBS resulted in a slight increase in Tb ion loss. Therefore, in various embodiments, the pretreatment time is preferably about 20 minutes, about 15 to 25 minutes, and less than about 30 minutes.

[0063] The Tb@PMSNs-Zwi-mPEG process was repeated after pretreatment. 5K Exudation analysis of nanoparticles in different buffer solutions. Compared with Tb@PMSNs-Zwi-mPEG without any pretreatment. 5K Exudation observed in nanoparticles Figure 10 (A and 10B), during the four-day period, there was almost no detectable Tb loss in the pretreated nanoparticles ( Figure 11 B), except that 0.1M Na2CO3 / NaHCO3 (pH 10.6) still showed 13% effusion ( Figure 10 Apart from (B). Unbound by theory, it is believed that ion loss in pretreated samples is due to the degradation of the mesoporous silica nanoparticle framework under alkaline conditions, a degradation mechanism supported by TEM analysis. Figure 10 C-10D). Therefore, in various embodiments, it is provided that allows the use of functionalized MSNs (e.g., Tb@PMSNs-Zwi-mPEG) provided herein. 5K Various buffer solution pretreatment methods are used in the mass flow cytometry study of nanoparticles.

[0064] Determination of mPEG 5K Grafting density is used to reduce or minimize NSB.

[0065] In various forms and embodiments, through mPEG 5K Changing the nanoparticles' effect on mPEG during the attachment step 5KAdjust mPEG by adjusting the w / w ratio (NP / PEG). 5K The grafting density was determined, and the degree of PEG grafting was quantified by TGA. Various PMSNs-Zwi-mPEGs were synthesized in these examples. 5K The characteristics of nanoparticles are summarized in Table 3 below.

[0066] Table 3. Physicochemical characterization of the series PMSN-Zwi-mPEG5K nanoparticles.

[0067]

[0068] Table 4. Summary of mPEG5K layer grafting density and its conformation determination parameters a .

[0069]

[0070] a The prediction of the PEG conformation is based on the Alexander de Gennes model (“Conformations of Polymers Attached to an Interface”, Macromolecules 1980, 13(5), 1069-1075).

[0071] b The mass loss was calculated from the TGA measurement. The experiment was repeated, and the second set of measurements is given in parentheses.

[0072] c It is calculated using equation (2).

[0073] d RF=αN³ / 5 Where α is the monomer length (0.35 nm for PEG), and N is the number of repeating PEG units (mPEG). 5K (108).

[0074] e The calculated average spacing between adjacent PEG anchor points, D=2(1 / π)¹ / ², where This represents the grafting density (number of chains per nm²).

[0075] f The calculated brush height, L=N(α) 5 / ³) / D² / ³.

[0076] mPEG in each sample 5K The weight percentage is calculated from the mass loss obtained from the TGA. Figure 12 (This is summarized in Table 4.) Reference Figure 12 A, the TGA curve reveals that with mPEG... 5K A clear pattern emerges regarding the weight loss as the amount added increases. (Reference) Figure 12 B. Weight loss initially rises to a peak and then gradually decreases, consistent with the established trend of grafting density. It is noteworthy that at an NP / PEG w / w ratio of 10:40, mPEG... 5K Both the weight loss percentage and grafting density reached their maximum values. Unbound by theory, it is assumed that PMSN nanoparticles can be considered as d for the purpose of current surface area calculations. The surface area of ​​the PMSN was calculated using a uniform sphere with a diameter of 45 nm and mesopores resembling straight cylinders. This value was then compared with the measured mPEG. 5K The loss amount and the number of nanoparticles in the solution are combined to calculate mPEG. 5K The grafting density (σ, number of chains per nm²) (Table 4).

[0077] The conformation of end-grafted PEG is based on the Alexander-de Gennes model (see "Conformations of Polymers Attached to an Interface", Macromolecules 1980, 13(5), 1069-1075), using PEG. 5KThe PEG chain length (RF) is determined by the Flory radius (RF), the distance (D) between adjacent PEG chain anchor points, and the length / thickness (L) of the grafted PEG layer. Based on these parameters, PEG chains can acquire two main conformations: "mushroom" or "brush". When D is greater than RF (RF / D < 1), the PEG chains on the nanoparticle surface are very flexible and cannot fully extend, resulting in a mushroom conformation with a relatively thin PEG layer. As the grafting density increases (RF / D > 1), the dense PEG chains generate higher osmotic pressure within the medium, inducing the PEG chains to extend outward, thus adopting a brush-like arrangement with a thicker layer. When the length of the PEG layer exceeds RF by at least twice (L / RF>2), a unique brush conformation is defined as a dense brush regime (see, for example, Damodaran, VB; Fee, CJ; Ruckh, T.; Popat, KC, "Conformational Studies of Covalently Grafted Poly(ethylene glycol) on Modified Solid Matrices Using X-ray Photoelectron Spectroscopy", Langmuir 2010, 26(10), 7299-7306). Figure 12 D illustrates an instance. Following this model, with mPEG 5K Grafting density increased from 0.27 to 0.54 chains per nm², and the various embodiments of MSNs presented herein exhibited a brush-like or densely packed brush-like conformation. The thickness (L) of the PEG layer ranged from 11 to 14 nm, which is consistent with that of mPEG. 5K The contribution of L to the hydrodynamic radius calculated in equation (1) h =17nm values ​​are comparable. These values ​​are consistent with the formation of dense brush-like PEG chains on the surface of nanoparticles.

[0078] In various embodiments, compositions and methods are provided for reducing and / or minimizing NSB of PEGylated PMSNs by selecting PEG surface coverage. Protein adsorption behavior was investigated by incubating nanoparticles (5 mg / mL in 1×PBS) with human serum albumin solution (HSA, 1 mg / mL in 1×PBS) at 37°C for 2 hours (Thermomixer, 500 rpm). The weight percentage (wt%) of HSA adsorbed on the nanoparticles (NPs) was determined using the Pierce™ BCA protein assay. Figure 13 A). For example Figure 12 As shown in Figure C, compared to the bare MSNs and PMSNs-Zwi samples, all polyethylene glycol-modified PMSNs significantly reduced HSA adsorption. In the polyethylene glycol-modified samples of this example, PMSN-Zwi-mPEG... 5K The nanoparticle (10:40) sample exhibited the lowest serum albumin adsorption. Unbound by theory, it is believed that the densest brush conformation is most effective in inhibiting nonspecific protein adsorption.

[0079] Antibodies and PMSNs-Zwi-PEG 5K - Coupling of N3 nanoparticles.

[0080] To test the ability of the NP-Ab conjugates disclosed herein as reporter molecules for mass spectrometry flow cytometry analysis, the bioconjugation efficacy of the antibody and nanoparticles was examined, and various conjugation conditions were determined. NP-Ab conjugation utilizes a copper-free click cycloaddition reaction of DBCO-azide. PMSNs containing azidides (PMSNs-Zwi-PEG) were synthesized. 5K -N3), and then conjugated it to DBCO-modified antibodies under various conjugation conditions. To test whether the bioconjugation conditions preserved antigen recognition ability, an anti-biotin antibody was used as a model, and biotin-Cy5 was used as the antigen target. Figure 14 Under ultraviolet-visible light (UV-vis) detection, antigen recognition will produce a characteristic peak at λ=649nm. As a control, mouse IgG2a,κ was used as an isotype control in parallel experiments.

[0081] In preparation of PMSNs-Zwi-mPEG 5K Under substantially similar reaction conditions, PMSNs-Zwi nanoparticles were reacted with N3-PEG. 5K - Silanes are reacted to prepare PMSNs-Zwi-PEG 5K -N3 nanoparticles. Figure 15 PMSNs-Zwi-PEG were displayed 5K TEM images of -N3 nanoparticles. These nanoparticles have characteristic dimensions d... =49.1±3.1nm and dh=79.9nm (PDI=0.095), similar to PMSNs-Zwi-mPEG. 5K Nanoparticles (see, for example, Figure 2 (C and 2D). These PMSNs-Zwi-PEG5K -N3 nanoparticles can be well redispersed in 1×PBS even after drying, and show good stability when stored in PBS at 4°C for up to 7 days. Figure 15 C). PMSNs-Zwi-PEG 5K The presence of active azide functional groups on the -N3 nanoparticles was determined by reacting them with DBCO-Cy5 at room temperature for 12 hours. After purification, the concentration of PMSNs-Zwi-PEG in 1×PBS was measured. 5K The UV-vis absorbance of -N3 nanoparticles can be clearly observed, with a characteristic absorbance peak at 649 nm attributed to the Cy5 group. Figure 15 D). Each PMSNs-Zwi-PEG 5K The number of azide groups in the -N3 nanoparticles was calculated to be approximately 630.

[0082] Purified anti-biotin antibodies and mouse IgG2a,κ isotype control antibodies were reacted with a 10-fold molar excess of DBCO-PEG4-NHS to introduce the DBCO functional group into the antibodies. After the reaction, the purified DBCO-Ab solution was measured by UV-Vis. The spectrum showed two characteristic absorbance peaks: DBCO at 309 nm and Ab at 280 nm. Figure 16 A). Using equation (S1), the number of DBCO groups per antibody is calculated to be approximately 4.

[0083] DBCO-Abs and PMSNs-Zwi-PEG were performed at an antibody-to-nanoparticle (Ab:NP) ratio of 22:1 or 50:1. 5K The reaction with -N3 nanoparticles was conducted using the following different conjugation conditions: (a) 37°C, 1 h; (b) 37°C, 4 h; (c) room temperature (RT, 25°C), 4 h; and (d) RT, 12 h. Excess unconjugated antibody was removed by multiple washes in a PALL centrifugal ultrafiltration system (300 kDa, 2,080 × g, 20 min). Figure 16 B shows the purified PMSNs-Zwi-PEG obtained after incubation at room temperature for 4 hours. 5K - DLS results for anti-biotin antibody conjugates. Compared to PMSNs-Zwi-PEG 5KWhen using -N3 nanoparticles, an increase of approximately 10 nm in the hydrodynamic diameter was observed in the NP-antibiotic Ab conjugate. This increase is in good agreement with the size of the antibiotic antibody (dh = 10.7 nm) as determined by DLS. The number of antibodies per nanoparticle was determined using the Micro™ BCA protein assay, with calibration curves plotted on [data missing]. Figure 13 In B, during a 4-hour bioconjugation process at room temperature, for example, at a feed ratio of 22:1, approximately 9 anti-biotin antibodies or 6 isotype control antibodies attached to each nanoparticle, while a feed ratio of 50:1 produced approximately 13 anti-biotin antibodies or 8 isotype control antibodies per nanoparticle. It was determined that for each bioconjugation condition, a higher Ab to NP feed ratio resulted in a greater number of antibodies attached to each NP.

[0084] To determine whether the reactivity of anti-biotin antibodies was retained after conjugation and purification, NP-anti-biotin antibody conjugates were incubated with a 4-fold excess of Cy5 biotin molecules in a ThermoMixer (Eppendorf, 500 rpm) at room temperature for 2 hours. After removing unbound Cy5 biotin by five cycles of centrifugation and ultrafiltration (Amicon, Ultra-0.5, 100 kDa, 4, 100 × g, 20 min, washed with H2O), UV-vis spectroscopy measurements were performed on eight different NP-anti-biotin antibody-Cy5 conjugates. Nanoparticles conjugated with isotype control antibodies obtained from various conjugation conditions were used as negative controls, undergoing the same Cy5 biotin incubation, centrifugation, ultrafiltration, and UV-vis detection process. A characteristic absorbance peak was observed at 649 nm for the NP-anti-biotin antibody-Cy5 conjugate, while no such peak was observed for the NP-control antibody-Cy5 conjugate. Figure 16 C (see illustration). This phenomenon remained constant under all eight different bioconjugation conditions, indicating that the anti-biotin antibody still functions and is able to bind to its target after conjugation and purification procedures.

[0085] The proportion of active antibodies was quantified by calculating the number of Cy5 molecules per antibody and used to evaluate the ability of various conjugation conditions to retain antibody function. The UV-vis spectrum of the precursor NP-antibiotin antibody conjugate was normalized to the spectrum of the NP-antibiotin antibody-Cy5 conjugate in the wavelength range of 400 to 550 nm, within which the dye Cy5 does not absorb. The corrected baseline was... Figure 16The value is shown as a dashed line in C. The difference between this corrected baseline and the absorption peak at 649 nm is considered a measure of the number of Cy5 biotin molecules captured by the anti-biotin antibodies. The number of Cy5 molecules per NP is calculated using equation (S2). Therefore, the Cy5-to-Ab ratio is determined by comparing the number of Cy5 molecules per NP with the number of antibodies per NP.

[0086] When the Ab to NP feed ratio was set to 22:1, a similar number of Cy5 biotin molecules were found per antibody under all conjugation conditions in this example. Figure 16 (D) In ​​contrast, a 50:1 ratio resulted in a higher Cy5 count per antibody. It is important to note that each IgG antibody has two identical antigen-binding sites (specific to biotin in our system). Nevertheless, no reaction conditions in this example allowed for the detection of such a high level of biotinylated Cy5 capture. However, Cy5 values ​​greater than 1.0 per antibody were determined, and a higher Ab-to-NP feed ratio and milder bioconjugation conditions favored obtaining antibodies that retained higher functionality.

[0087] The present invention and its various forms and embodiments will be described in more detail with reference to the following examples, but it should be understood that the present invention, forms and embodiments are not to be considered as limited thereto.

[0088] Example Example 1 - Instruments and Equipment.

[0089] Transmission electron microscopy (TEM): Nanoparticle samples were dispersed in H₂O and drop-cast onto Formvar / Carbon 200-mesh grids. All TEM images were acquired on a Hitachi HT7700 TEM instrument operating at 80 kV. Particle size was measured using Image-J, and size distribution histograms were plotted using Origin.

[0090] Dynamic Light Scattering (DLS) Measurement: DLS measurements were performed at 25°C on a Malvern Zetasizer Nano ZS instrument. Aqueous samples were placed in ZEN0040 disposable plastic micro cuvettes. Samples were dispersed in water or 1×PBS buffer at a concentration of approximately 0.1 mg / mL. Particle size measurements were performed at a scattering angle of 173°. Z-average hydrodynamic diameters (dh), polydispersities (PDI), and intensity distribution maps were obtained using the instrument's built-in software.

[0091] Fourier-Transform Infrared (FTIR) Spectrometer: All FTIR spectra were collected using a PerkinElmer Spectrum Two ATR-FTIR spectrometer equipped with a diamond crystal polarization accessory. Each sample was collected at 4.0 cm⁻¹. - ¹ 64 scans were performed at a resolution of 1.

[0092] Thermogravimetric analysis (TGA): Quantitative determination of functional groups and polymers bound to the MSN surface was performed in nitrogen at a flow rate of 100 mL / min using an SDTQ600 thermogravimetric analyzer. The temperature was raised to 100 °C and held constant for 30 minutes to ensure desorption of adsorbed water. Finally, the temperature was raised to 800 °C at a rate of 10 °C / min.

[0093] Nitrogen adsorption / desorption isotherms: Nitrogen adsorption / desorption isotherms were measured on a Quantachrome Instruments Autosorb-iQ (Boynton Beach, Florida, USA) equipped with ultra-high purity gases. The samples were degassed in a gradient manner for a total of 12 hours. The surface area was determined from the adsorption isotherm using the Brunauer-Emmett-Teller (BET) method (see Konry, T.; Smolina, I.; Yamush, JM; Irimia, D.; Yamush, ML, “Ultrasensitive Detection of Low-Abundance Surface-Marker Protein Using Isothermal Rolling Circle Amplification in a Microfluidic Nanoliter Platform”, Small 2011, 7(3), 395-400), while the pore size and pore volume were calculated from the desorption branch of the isotherm using the Barrett-Joyner-Halenda (BJH) method (see Behbehani, GK, “Applications of mass cytometry in clinical edicine: the promise and perils of clinical CyTOF:, Clinics inlaboratory medicine 2017, 37(4), 945-964).

[0094] Nuclear Magnetic Resonance (NMR) spectroscopy:¹H NMR measurements were performed on a 400MHz Agilent DD2 NMR spectrometer or a 600MHz Agilent DD2 NMR spectrometer.

[0095] Inductively Coupled Plasma Mass Spectrometry (ICP-MS): All measurements were performed using a Thermo Scientifici CAPQICP-MS system. A 2 v / v% HNO3 solution was prepared in ultrapure water from high-purity HNO3. A series of calibration standards with concentrations of 0.1, 1, 10, 20, and 40 ppb were prepared from the standard solution in 2 v / v% HNO3. An internal standard of approximately 20 ppb was used. All samples were diluted 50, 100, and 500 times to obtain the optimal concentration falling within the calibration concentrations. Each measurement was performed in triplicate, and the final ppb value of the target element was calculated as the average of all measurements at different dilutions.

[0096] The ZetaView Nanoparticle Tracking Analyzer (NTA) uses a diluted sample of 100 nm polystyrene microspheres (Nanosphere™, Thermo Fisher Scientific) as a standard solution to calibrate the instrument. A sample solution (1.5–2 mL) with a known concentration (mg / mL) is injected into the instrument for measurement. The measured nanoparticle concentration is given in particles / mL, and these values ​​can be converted to particles / gram using a known mass concentration.

[0097] UV-Vis Spectrophotometer: UV-Vis measurements were performed on a BioTek Epoch 2 microplate spectrophotometer. All sample dispersions were placed in BrandTech™ BRAND™ disposable cuvettes with a 1 cm path length or Thermo Scientific Pierce™ 96-well plates.

[0098] Example 2 - Materials Tetraethoxysilane (TEOS), triethanolamine (TEA), triethylamine, hexadecyltrimethylammonium chloride (CTAC, 25wt%), (N,N-dimethylaminopropyl)trimethoxysilane (DMASi), 1,3-propanesulfonic acid lactone, sodium azide, dibenzocyclooctyne-PEG4-N-hydroxysuccinimide ester (DBCO-PEG4-NHS, ≥90%), albumin from human serum (HSA, protease-free, ≥96%), and metal salts with a purity ≥99.99% (trace metal standard), including lanthanum(III) chloride heptahydrate (LaCl3·7H2O), cerium(III) chloride heptahydrate (CeCl3·7H2O), praseodymium chloride hexahydrate (PrCl3·6H2O), neodymium(III) chloride hexahydrate (NdCl3·6H2O), and samarium(III) chloride hexahydrate. The following compounds were purchased from Sigma-Aldrich (CA): europium(III) chloride hexahydrate (SmCl3·6H2O), gadolinium(III) chloride hexahydrate (GdCl3·6H2O), terbium(III) chloride hexahydrate (TbCl3·6H2O), holmium(III) chloride hexahydrate (HoCl3·6H2O), erbium(III) nitrate pentahydrate (Er(NO3)3·5H2O), thulium(III) chloride hexahydrate (TmCl3·6H2O), ytterbium(III) nitrate hydrate (Yb(NO3)3·5H2O), lutetium(III) chloride hexahydrate (LuCl3·6H2O), single-element standard solutions (Ho, Tb, and Ta), and multi-element standard solutions for inductively coupled plasma mass spectrometry (ICP-MS) calibration. Silane-PEG 5K -Methoxy group (M5000 g / mol) was purchased from JenKem Technology (USA). Silane-PEG 5K - The azide was purchased from Biopharma PEG (Watertown, MA, USA). 3-Methoxy(polyethyleneoxy)6-9-propyltrimethoxysilane (3-Methoxy(polyethyleneoxy)) 6-9propyltrimethoxy silane (PEG6-9silane, 459-591 g / mol) was purchased from Gelest (Morrisville, PA, USA). Phosphate-buffered saline (1×PBS solution, 0.137 M NaCl, 0.0027 M KCl, and 0.0119 M phosphate at pH 7.4, Fisher BioReagents), high-purity nitric acid (trace metal grade, 67-70%, Optima™) for ICP-MS, the Pierce™ BCA Protein Assay Kit, and the Micro™ BCA Protein Assay Kit were purchased from Fisher Scientific. Cy5 biotin was purchased from Click Chemistry Tools (Scottsdale, AZ, USA). Purified anti-biotin antibody (clone, 1D4-C5) and isotype control antibody (purified mouse IgG2a,κ, clone MOPC-173) were purchased from BioLegend. Antibodies were purified by centrifugation and ultrafiltration with 1×PBS (Amicon, Ultra-0.5, 10kDa) to remove sodium azide before use. All other chemicals were used as is without further purification.

[0099] Example 3 - Synthesis of PEG6-9 modified mesoporous silica nanoparticles (PMSNs).

[0100] The synthesis of PMSNs was as follows. Hexadecyltrimethylammonium chloride (CTAC, 2 g) and triethanolamine (TEA, 0.8 g) were dissolved in water (18.2 MΩ, 20 mL) and magnetically stirred at room temperature for 1 hour. The reaction mixture was heated to 95 °C and stirred for another 1 hour. Tetraethoxysilane (TEOS, 1.5 mL) was injected into the reaction mixture at a rate of 150 μL / min using a syringe pump. After the injection of TEOS, the mixture was stirred for 10 minutes, and then 3-[methoxy(polyvinyloxy)6-9]propyltrimethoxysilane (PEG) was injected at a rate of 65 μL / min. 6-9 Silane (650 μL) was injected into the solution. The mixture was then stirred at 95 °C for 30 minutes. After cooling the reaction mixture to room temperature, the resulting CTAC-containing PMSNs were collected using ethanol through three cycles of sedimentation-redispersion (45,000 × g, 45 min).

[0101] To remove the CTAC template, the nanoparticles were dispersed in 50 mL of acidic ethanol solution (concentrated hydrochloric acid at 10 v / v% in ethanol) and refluxed at 85 °C for 12 h. This extraction was performed twice to ensure complete removal of CTAC. Finally, the PMSNs were collected with ethanol through three sedimentation-redispersion cycles (45,000 × g, 45 min) and then dried overnight in a vacuum oven.

[0102] Example 4 - Synthesis of zwitterionic modified mesoporous silica nanoparticles (PMSNs-Zwi).

[0103] For the synthesis of zwitterionic sulfobetaine silane (SBS), a 25 mL round-bottom flask equipped with a magnetic stirrer was sealed with a rubber diaphragm and purified with N2 for 30 min. Under N2 atmosphere, (N,N-dimethylaminopropyl)trimethoxysilane (DMASi, 2.07 g, 10 mmol), 1,3-propanesulfonyl lactone (1.34 g, 11 mmol), and anhydrous acetone (10 mL) were added to the reaction flask using a syringe. The reaction was stirred vigorously at room temperature for 6 h. The white precipitate was centrifuged (4,000 rpm, 10 min) and washed three times with anhydrous acetone to remove unreacted reagents. The final product was dried under vacuum at 30 °C for 24 h and stored under N2. The yields were between 60% and 75%. The ¹H NMR and ¹³C NMR spectra of the product were obtained using a 400 MHz Agilent DD2 NMR spectrometer and are presented in [image missing]. Figure 3 In B and 3C.

[0104] The PMSNs sample (50 mg) was redispersed in H2O (4 mL) under sonication, producing a clear, light blue solution. The solution was purged with N2 for at least 15 minutes. Zwitterionic sulfobetaine silane (50 mg) was dissolved in H2O (1 mL) and then added dropwise to the PMSN solution. The pH was adjusted to approximately 9 using 28% ammonia. The mixture was stirred at 80 °C for 24 hours. The resulting nanoparticles were washed with excess H2O in each cycle through three sedimentation-redispersion cycles (45,000 × g, 45 min) and then freeze-dried.

[0105] Example 5 - PEG 5K Silane-modified mesoporous silica nanoparticles (PMSNs-Zwi-PEG) 5K Synthesis of ).

[0106] Using silane-PEG under similar conditions 5K -Methoxy (mPEG) 5K ) or silane-PEG 5K - Azide (N3-PEG) 5KPolyethylene glycol-modified PMSNs-Zwi nanoparticles with different functional groups were prepared. Typically, PMSNs-Zwi (10 mg) was added to a two-necked round-bottom flask (25 mL) and suspended in anhydrous toluene (5 mL). The resulting suspension was purged with N2 for 15 min, and mPEG in 1 mL of anhydrous toluene was removed. 5K The solution was added dropwise to the suspension. The reactants were stirred at 110°C for 24 hours. After cooling, the nanoparticles were collected by centrifugation (45,000 × g, 45 min) and washed three times with ethanol to remove unreacted mPEG. 5K Silane. The final PMSNs-Zwi-mPEG 5K Suspended in water and freeze-dried to obtain a free-flowing powder. This was achieved by processing mPEG under the same conditions. 5K Adjusting the dosage from 10 mg to 80 mg can yield different mPEG levels. 5K Samples with grafting density. Additionally, N3-PEG with a PEG-silane mass ratio of 10:40 was obtained by using nanoparticles. 5K Silane was used to prepare polyethylene glycolated MSNs (PMSNs-Zwi-PEG) with azide functional groups under the same conditions. 5K -N3).

[0107] Example 6 - Lanthanide loading and efflux analysis.

[0108] In a typical loading experiment, 10 mg of PMSNs-Zwi-mPEG was loaded. 5K Nanoparticles were dispersed in H2O (2 mL) under ultrasonic treatment. TbCl3·6H2O (47 mg, 10 mg / mL Tb) was added to the suspension, and the mixture was stirred at room temperature for 12 hours. Tb-loaded PMSNs-Zwi-mPEG was obtained by centrifugation (45,000 × g, 45 min). 5K (Tb@PMSNs-Zwi-mPEG) 5K Nanoparticles were collected and washed twice with water. After the final centrifugation, 20 mL of 1×PBS buffer was added to precipitate Tb3+ as TbPO4 within the pores. The Tb-containing nanoparticles were then immersed in 1×PBS buffer at room temperature for 20, 40, 60, or 120 minutes without stirring. The nanoparticles were then washed with H2O through three sedimentation-redispersion cycles (45,000×g, 45 minutes). After freeze-drying, Tb@PMSNs-Zwi-mPEG was used. 5K Nanoparticles were dispersed in 2v / v%HNO3, and the amount of Tb in the nanoparticles was determined by ICP-MS analysis.

[0109] For multi-element loading, PMSNs-Zwi-mPEG in water were prepared. 5K Samples (2.5 mg, 0.5 mg / mL, 5.0 mL). A series of lanthanide salts were added sequentially to the solutions, with each lanthanide salt added in an amount sufficient to achieve a final concentration of 1 mg / mL. 3+ Er 3+ and Yb 3+ It is added in the form of nitrates, while La 3+ Ce 3+ 、Nd 3+ 、Sm 3+ Eu 3+ Gd 3+ 、Tb 3+ Ho 3+ Tm 3+ and Lu 3+ It is added in the form of chloride salt. The mixture is stirred at room temperature for 24 hours.

[0110] Using Tb@PMSNs-Zwi-mPEG 5K The efflux of Ln ions from lanthanide-loaded nanoparticles was tested. For each measurement, a nanoparticle sample (3 mg) was dispersed in a buffer solution (5 mL) and vigorously stirred at room temperature. Different buffers, including 0.1 M sodium carbonate / sodium bicarbonate buffer (Na₂CO₃ / NaHCO₃, pH 10.6), 0.1 M MES buffer (pH 5.5), 0.02 M Bis-tris buffer (pH 6.5), 0.01 M HEPES buffer (pH 7.4), H₂O, and 1×PBS buffer, were used for these efflux tests. Aliquots (0.5 mL) were then periodically removed and transferred to 1.5 mL centrifuge tubes over 7 days. Each sample was centrifuged at 45,000×g for 45 min. After centrifugation, the supernatant was diluted 50-fold with 2 v / v % HNO₃ for ICP-MS analysis.

[0111] Example 7 - Serum protein adsorption analysis.

[0112] Protein adsorption experiments were performed using protease-free human serum albumin (HSA) at 37°C for 2 hours. The nanoparticle sample (1.000 ± 0.040 mg, weighed on a microbalance) was redispersed in 1×PBS buffer (200 μL) under sonication. Then, 1×PBS buffer solution of HSA (200 μL, 1 mg / mL) was added. The mixture was incubated in an Eppendorf ThermoMixer at 37°C with shaking at 500 rpm for 2 hours. After incubation, loosely bound proteins were removed using an Amicon Ultra-0.5 centrifuge ultrafilter (100 kDa, 10,000 rpm, 10 min), and the sample was washed five times with fresh 1×PBS buffer. The amount of adsorbed HSA protein was determined using the Pierce™ BCA assay (Thermo Fisher Scientific) at λ = 562 nm. The supernatant was collected after each centrifugation and analyzed by UV-vis to ensure that all loosely bound proteins were removed.

[0113] Example 8 - Antibodies and PMSNs-Zwi-PEG 5K -N3 nanoparticle coupling; preparation of DBCO-functionalized antibody (DBCO-Ab).

[0114] Following the same protocol, two antibodies were bioconjugated using the same method: an anti-biotin antibody and a control antibody. As an example, the antibody stock solution (100 μL, 1.6 mg / mL in 1×PBS buffer, pH 7.4) was incubated with the DBCO-PEG4-NHS stock solution (1.1 μL, 10 mM in anhydrous DMSO, 10 molar equivalents) at room temperature for 1 hour. Unreacted DBCO-PEG4-NHS was removed by centrifugation and ultrafiltration (Amicon, Ultra-0.5, 10 kDa) at 12,000×g for 10 minutes, followed by washing three times with 1×PBS buffer. The DBCO-Ab solution was then diluted to a total volume of 200 μL using 1×PBS buffer (pH 7.4). Antibody concentration and the number of DBCO groups per antibody were determined by UV-vis measurement. The number of DBCO groups per antibody was calculated according to the following equation:

[0115] Where A(309nm) and A(280nm) are the absorbance values ​​of the DBCO-Ab solution at λ=309nm and λ=280nm, respectively. ɛ(DBCO) and ɛ(Ab) are the absorbance values ​​of DBCO (12,000M) at λ=309nm and λ=280nm, respectively. - ¹·cm - ¹) and antibodies (210,000M) - ¹·cm- ¹) is the molar extinction coefficient. 1.089 is the DBCO correction factor at 280 nm.

[0116] Example 9 - DBCO-Abs and PMSNs - Zwi-PEG 5K -N3 nanoparticle bioconjugation (NP-Ab).

[0117] For bioconjugation, PMSN-Zwi-PEG is used. 5K -N3 nanoparticles were coupled to DBCO-Ab via click chemistry. The coupling reaction was carried out under several different conditions: 1 hour at 37°C, 4 hours at 37°C, 4 hours at room temperature, or 12 hours at room temperature. Different amounts of DBCO-Ab solution were mixed with the nanoparticle solution (5 mg / mL, 100 μL) to adjust the Ab to NP ratio to 22:1 or 50:1. In a typical reaction with an Ab to NP ratio of 50:1, DBCO-Ab (45 μL, 0.4 mg / mL) in 1×PBS buffer was coupled with PMSN-PEG in 1×PBS buffer. 5K -N3 nanoparticles (100 μL, 5 mg / mL) were mixed, with DBCO-Ab concentration in the resulting mixture at approximately 0.86 μM. The coupling reaction was carried out at 37 °C for 4 h using a Thermo Mixer (Eppendorf, 500 rpm), followed by quenching with the addition of sodium azide (8 μL, 1 wt% in deionized water, room temperature, 1 h). The NP-Ab conjugate solution was ultrafiltered by centrifugation at 2,080 × g (PALL, 300 kDa) for 20 min and washed four times with 1 × PBS buffer to remove unreacted DBCO-Ab and sodium azide. Finally, additional PBS buffer (pH 7.4) was added to adjust the final volume to 100 μL. 15 μL of the purified conjugate solution was then diluted 10-fold for Micro™ BCA analysis to quantify the antibody count for each NP.

[0118] Example 10 - Using Cy5 biotin-labeled NP-Ab and quantitative active antibody.

[0119] NP-Ab was labeled with Cy5 biotin (NP-Ab-Cy5). Based on the number of antibodies on each nanoparticle, a 4-fold excess of Cy5 biotin was mixed with the NP-Ab conjugate solution. In a typical reaction, the NP-Ab solution (60 μL) was mixed with the Cy5 biotin solution (0.85 μL, 0.2 mg / mL in H2O). The mixture was vortexed for 2 h at room temperature using a Thermo Mixer (Eppendorf, 500 rpm). After the reaction, the mixture was purified by ultrafiltration (Amicon, Ultra-0.5, 100 kDa) at 4,100 × g for 15 min and washed five times with H2O. Additional H2O was added to reach a final volume of 60 μL. The number of Cy5 molecules per nanoparticle was quantified using UV-vis spectroscopy.

[0120] Cy5 was quantified for each antibody. Each NP-Ab solution was measured by UV-vis prior to incubation with Cy5 biotin. The NP-Ab spectra from 400 nm to 550 nm were normalized to the corresponding NP-Ab-Cy5 spectra using the least squares method. The normalized NP-Ab spectra were used as the model baseline. The absolute absorbance value (A649) of Cy5 was obtained by subtracting the absorbance value of the normalized NP-Ab spectrum at λ=649 nm from the absorbance value of the corresponding NP-Ab-Cy5 spectrum at λ=649 nm. The number of Cy5 groups per nanoparticle was calculated according to the following equation:

[0121] Where N AV It is Avogadro's number, and ɛ(Cy5) is the molar extinction coefficient of Cy5 (250,000 M). - ¹·cm - ¹), cNTA(NP) is the NP concentration (particle number / mL) obtained from NTA. Factor 10 - ³ is used to convert mL to L. The Cy5 count per antibody is calculated as the ratio of the Cy5 count per nanoparticle to the antibody count per nanoparticle.

[0122] Example 11 - Characterization of surface modification by ¹H NMR The surface modifications of MSNs were also examined by ¹H NMR. Figure 8 PMSNs, PMSNs-Zwi, and PMSNs-Zwi-mPEG were compared. 5KSuspension spectroscopy in D2O. In the ¹H NMR spectra of PMSNs, a hydrogen-related peak at 3.5–3.6 ppm was observed, which is attributed to the PEG incorporated into the nanoparticles during the first synthetic step. 6-9 Silane. After SBS modification, PMSNs-Zwi retained the resonance signal corresponding to the PEG unit and, with Figure 3 The SBS spectra shown are very similar. This is especially true after the introduction of PEG. 5K After polymerization, the signal area attributed to the -CH2-CH2O- hydrogen atoms significantly increased, while the SBS peak remained visible. These results clearly demonstrate the simultaneous presence of SBS and mPEG in multifunctional nanoparticles. 5K Silane.

[0123] Example 12 - Estimating mPEG 5K Derivation of the Graft Density Equation

[0124] Where σ PEG / nm² This represents the mPEG per nm² of PMSNs surface area. 5K The grafting density of the chain, ρ represents the density of silica (1.85-2.2 g / cm³) 4-5, and β is the mPEG in the sample obtained through TGA. 5K The weight ratio (in mg / g), NAV is Avogadro's constant, d (nm) is the average diameter measured from the TEM image, and M is mPEG. 5K The molecular weight is 5000 g / mol, and Vp is the pore volume (0.96 cm³ / g) obtained by nitrogen adsorption / desorption analysis using the BJH method. Factor 10 - ² 4 It is used to convert m² units to nm².

[0125] To determine the surface area available for PEG modification, it is necessary to start from the total surface area of ​​the sphere (S sph Subtract the value attributed to mesopore openings (S) from the original text. po e The void surface area. Therefore,

[0126] Where V sph The volume of the particle is assumed to be spheres and calculated as V. sph =4 / 3π(d / 2)³, S sph It is the total surface area of ​​a single nanoparticle, calculated as S. sph =πd². Therefore, equation (S3) can be transformed into:

[0127] S po e This represents a void surface, which can be modeled as the base of a cylinder with a diameter (d). po e The pore size is obtained from BJH data. Therefore, S po e By using d po e Multiply by the number of pores per nanoparticle (n) po e To calculate, n is used to calculate, where n is used to calculate. po e Equal to twice the number of bases of each cylinder (2ncylinder):

[0128] Number of porous cylinders (n) cylinder ) is achieved by adjusting the pore volume (V) of individual nanoparticles. po e ρV sph Divide by the volume of the cylinder (V) cylinder It is calculated using ) where V cylinder The height is estimated by using the diameter (d) of the nanoparticles as the reference value:

[0129] Combining equations (S5) and (S6) and substituting these items into equation (S4), we obtain equation (2). Based on this, the mPEG on the outer surface of the mesoporous silica nanoparticles is calculated. 5K Estimate the density.

[0130] Example 13-mPEG 5K Conformation determination The Florey radius (RF), grafting distance (D), and mPEG were determined using the following equations. 5K Layer thickness (L):

[0131] Where α is the monomer length (0.35 nm for PEG), and N is the number of repeating PEG units (mPEG used in this study). 5K (108), and α is the measured mPEG. 5K Grafting density (number of chains per nm²).

[0132] Although the invention has been described in detail and referenced to specific examples, styles and embodiments, it will be apparent to those skilled in the art that various changes and modifications may be made therein without departing from its spirit and scope.

[0133] Declarations regarding incorporation and variants via reference All references cited in this application, such as patent documents, including issued or granted patents or equivalents and patent application publications, as well as non-patent documents or other sources, are incorporated herein by reference in their entirety as if they were cited separately. None of them are acknowledged as prior art.

[0134] When a set of substituents is disclosed herein, it should be understood that all individual members of these groups, as well as all subgroups and categories, that can be formed using the substituents are disclosed separately. When the Markush group or other groupings are used herein, all individual members of the group and all possible combinations and subcombinations are intended to be included individually in the disclosure. As used herein, "and / or" means that one, all, or any combination of items separated by "and / or" in the list is included in the list; for example, "1, 2 and / or 3" is equivalent to "1, 2, 3, 1 and 2, 1 and 3, 2 and 3, or 1, 2 and 3".

[0135] Unless otherwise stated, every formulation or combination of ingredients described or illustrated is applicable to the practice of this invention. Specific names of materials are intended as examples, as those skilled in the art know that the same material can be named under different names. It is understood that, in practicing this invention, methods, apparatus components, starting materials, and synthetic methods other than those specifically illustrated can be employed without excessive experimentation. All functional equivalents known in the art of any such methods, apparatus components, starting materials, and synthetic methods are intended to be included in this invention. Whenever a range is given in the specification, such as a temperature range, time range, or composition range, all intermediate ranges and sub-ranges, as well as all individual values ​​contained within a given range, are intended to be included in this disclosure. It should be understood that, unless the context clearly specifies otherwise, each intermediate value (accurate to one-tenth of the lower limit unit) between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any specified value or intervention value contained within the specified range and any other specified value or intervention value within the specified range is covered. The upper and lower limits of these smaller ranges may be independently included or excluded from the range, and each range that includes, does not include, or includes both limitations is also included in the invention, but is subject to any explicitly excluded limitations in the defined range. When the defined range includes one or two limitations, the range that excludes any or both of these included limitations is also included.

[0136] As used herein, "comprising" is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended, not excluding additional, unlisted components or method steps. As used herein, "consisting of" excludes any component, step, or ingredient not specified in the elements of the claim. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the essential and novel features of the claim. Any reference to the term "comprising," particularly when describing the components of a composition, a method, or an apparatus, is to be understood to cover those compositions, methods, or apparatuses that consist primarily of or are composed of the listed components or elements, with the option to add other components or elements. The invention exemplarily described herein can be practiced without any components, multiple components, limitations, or multiple limitations not specifically disclosed herein.

[0137] As used herein and in the appended claims, the singular forms "a," "an," and "the" include a plurality of indicators unless the context clearly indicates otherwise. Thus, for example, a reference to "a method" includes a plurality of such methods, a reference to "nanoparticles" includes a reference to one or more nanoparticles and their equivalents known to those skilled in the art, and so on. The terms and expressions used are used as descriptive rather than limiting terms, and their use is not intended to exclude any equivalents of the shown and described features or portions thereof, but it should be recognized that various modifications are possible within the scope of the claimed invention. Therefore, it should be understood that although the invention has been specifically disclosed through preferred embodiments and optional features, modifications and variations of the concepts disclosed herein are possible to those skilled in the art, and such modifications and variations are considered to be within the scope of the invention as defined by the appended claims.

Claims

1. A composition comprising functionalized mesoporous silica nanoparticles (MSN), characterized in that, The functionalized mesoporous silica nanoparticles comprise: Mesoporous silica nanoparticles (MSN); At least one zwitterionic silane; and At least one polyethylene glycol (PEG) functionalized silane.

2. The composition according to claim 1, characterized in that, The at least one zwitterionic silane is a silane containing a zwitterionic betaine group.

3. The composition according to claim 1, characterized in that, The at least one zwitterionic silane is a zwitterionic sulfobetaine silane.

4. The composition according to claim 3, characterized in that, The at least one polyethylene glycol (PEG) functionalized silane is PEG 5k Silane.

5. The composition according to claim 1, characterized in that, The functionalized MSN further contains lanthanide ions.

6. The composition according to claim 5, characterized in that, The lanthanide ions are selected from the group consisting of La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu and combinations thereof.

7. The composition according to claim 1, characterized in that, The functionalized MSN further contains Tb ions.

8. The composition according to claim 6, characterized in that, Each of these functionalized MSNs contains at least approximately 1 × 10 4 One lanthanide ion.

9. The composition according to claim 6, characterized in that, Each of these functionalized MSNs contains at least approximately 5 × 10 4 One lanthanide ion.

10. The composition according to claim 4, characterized in that, The PEG 5K -Silane is an azide-PEG 5K -Silane.

11. The composition according to claim 1, characterized in that, The functionalized MSN is coupled with dibenzocyclooctylene-functionalized anti-biotin antibody (DBCO-anti-biotin Abs).

12. The composition according to claim 2, characterized in that, The PEG 5K -Silane is an azide-PEG conjugated with dibenzocyclooctylene-functionalized anti-biotin antibodies (DBCO-anti-biotin Abs). 5K -Silane.

13. A mixture of functionalized MSNs, characterized in that, The mixture comprises multiple functionalized MSNs, each of which contains: Mesoporous silica nanoparticles (MSN); At least one zwitterionic silane attached to the MSN; At least one polyethylene glycol (PEG) functionalized silane attached to the MSN; and At least one lanthanide ion attached to the MSN.

14. The mixture according to claim 13, characterized in that, The at least one zwitterionic silane is a zwitterionic sulfobetaine silane.

15. The mixture as claimed in claim 13, characterized in that, The at least one polyethylene glycol (PEG) functionalized silane is PEG 5K Silane.

16. The mixture as claimed in claim 15, characterized in that, The PEG 5k -Silane is an azide-PEG 5k -Silane.

17. The mixture as claimed in claim 13, characterized in that, The lanthanide ions are selected from the group consisting of La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu and their combinations.

18. The mixture as claimed in claim 16, characterized in that, Each of these functionalized MSNs contains at least approximately 1 × 10 4 One lanthanide ion.

19. The mixture as claimed in claim 13, characterized in that, At least a portion of the MSN further comprises at least one antibody attached to at least one of the polyethylene glycol (PEG) functionalized silanes.

20. The mixture according to claim 13, characterized in that, The mixture was freeze-dried.

21. A method for manufacturing functionalized mesoporous silica nanoparticle reagents, characterized in that, Includes the following steps: A mixture of functionalized mesoporous silica nanoparticles is provided, each of the functionalized mesoporous silica nanoparticles (MSN) comprising a mesoporous silica nanoparticle (MSN), at least one zwitterionic silane attached to the MSN, at least one polyethylene glycol (PEG) functionalized silane attached to the MSN, and at least one lanthanide ion attached to the MSN. The mixture was contacted with phosphate-buffered saline (PBS) having a pH in the range of about 7.3 to 7.5, a NaCl concentration of about 0.14 M, a KCl concentration of about 0.003 M, and a total phosphate concentration of about 0.02 M; and The mixture of PBS and the functionalized MSN is reacted at a temperature ranging from about 20°C to about 25°C for about 10 minutes to about 40 minutes to produce the functionalized mesoporous silica nanoparticle reagent.

22. The method as described in claim 21, characterized in that, The reaction time is between approximately 15 minutes and approximately 25 minutes.

23. The method as described in claim 21, characterized in that, The at least one zwitterionic silane is a zwitterionic sulfobetaine silane.

24. The method as described in claim 21, characterized in that, The at least one polyethylene glycol (PEG) functionalized silane is PEG 5K Silane.

25. The method as described in claim 21, characterized in that, The PEG5K-silane is azide-PEG. 5K -Silane.

26. The method as described in claim 21, characterized in that, The lanthanide ions are selected from the group consisting of La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu and their combinations.

27. The method as described in claim 21, characterized in that, Each of these functionalized MSNs contains at least approximately 1 × 10 4 One lanthanide ion.

28. The method as described in claim 21, characterized in that, The method further includes the step of lyophilizing the functionalized mesoporous silica nanoparticle reagent to produce lyophilized functionalized mesoporous silica nanoparticle reagent.

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