HK40029552B - Functionalized nanoparticles and methods of making and using same - Google Patents

Description

Functionalized nanoparticles and methods of making and using the same

Cross Reference to Related Applications

This application claims priority to U.S. provisional application No. 62/508,703, filed on 19/5/2017, the disclosure of which is incorporated herein by reference.

Statement regarding federally sponsored research

The invention was made with government support granted grant number CA199081 by the national institutes of health. The government has certain rights in the invention.

Technical Field

The present disclosure relates generally to surface functionalized nanoparticles and methods of making and using the same. More specifically, the present disclosure relates to surface functionalized silica and aluminosilicate nanoparticles.

Background

Although, Silica Nanoparticles (SNPs) are attractive for potential therapeutic/diagnostic applications due to their large surface area, inertness, and high biocompatibility. However, most SNPs have a size >10 nm.

Particles larger than 12nm are not efficiently cleared from the body and also disadvantageously distribute in the liver and other organs/tissues, potentially exposing these tissues to toxic elements (especially if these SNPs larger than 10nm have drug and/or radioactive modifications). Particles with a diameter of about 8nm can stay in the body for about 1 day, about 3-5 days at 10-11nm, but if larger than 12nm, they are not or very slowly cleared.

At present, ultra-small inorganic nanoparticles are rapidly receiving attention as nano-drugs for cancer diagnosis and treatment. Due to their multifunctionality and multivalent effect, some organic nanomedicines have been more competitive with conventional chemotherapeutic drugs. Inorganic nanoparticles also diversify the building elements of nano-drugs and may have several advantages associated with their inherent physical properties and lower manufacturing costs. The safe transfer of nanoparticles from the laboratory to the clinic requires overcoming a number of significant scientific and legal hurdles. The most important criteria are a good biodistribution and its time evolution (pharmacokinetic, PK) profile. The size threshold for renal clearance is below 10 nm. To date, only a few inorganic nanoparticle platforms have been synthesized, with dimensions less than 10nm, allowing effective renal clearance to be achieved. Among these, only polyethylene glycol coated (pegylated) fluorescent core-shell Silica Nanoparticles (SNPs) of size <10nm, called Cornell dots or C-dots for short, were approved by the us Food and Drug Administration (FDA) and could be used for the first time as research-type new drugs (IND) in human clinical trials. Although the first clinical trial results for melanoma patients are promising, there are still some synthetic difficulties for this fluorescent organic-inorganic hybrid SNP of less than 10nm in size.

First, all previous efforts at C-site SNP synthesis followed improvedA process which uses an alcohol as a solvent. However, for materials used in biological or clinical applications, water is preferred as the reaction medium. This will greatly simplify the synthesis and cleaning process, reduce volatile waste, thereby greatly speeding up the production of granules and making them more cost effective.Furthermore, althoughThe method is widely used to produce SNPs with diameters from tens of nanometers to the micron scale, but due to the reaction kinetics limitations in alcohols, particle sizes of 10nm and below are at the limit of the synthetic method for size control.

Secondly, covalent coating of the silica particle surface with PEG can be troublesome, since loss of surface charge during pegylation may lead to particle aggregation or at least to an expanded particle size distribution. For ultra-small particles, this effect is more pronounced due to the increase in surface energy of the particles, thus limiting the monodispersity and size control capability of the particles.

Third, since the silica surface is negatively charged above its isoelectric point, pH 2-3, the covalent encapsulation of the negatively charged group of silane-conjugated organic fluorescent dyes in SNPs is inefficient under the influence of electrostatic repulsion between the silica and the fluorophore. This is particularly true for Near Infrared (NIR) emitting dyes that are most needed for imaging applications in living tissue. NIR dyes have a large delocalized pi-electron system and are generally required to form multiple negatively charged functional groups (e.g., sulfates) around their periphery for solubility in water. The low encapsulation efficiency of these dyes is a problem because they typically cost $200- $ 300/mg and reuse of commonly used silane-dye conjugates after initial synthesis is difficult.

Finally, for fluorescent SNPs and core-shell SNPs with sizes less than 10nm, no other inorganic element composition has been reported except for silica. In particular, it would be desirable to find compositions that result in an organic dye environment with increased rigidity, since the increase in rigidity is directly related to the increase in fluorescence yield of each dye resulting from the decrease in nonradiative rate. Silica compositions derived from aluminium alkoxides (aluminium alkoxides) are of particular interest here as additives, as they are known to be hardening components in alkoxysilane derived silica, and alumina is an approved adjuvant, which can be added to large-dose (high-volume) vaccines for intramuscular and subcutaneous injection.

These difficulties suggest that the original fluorescent core-shell SNP (C-site) synthesis approach is being re-investigated in order to systematically develop a water-based approach to produce <10nm organic-inorganic hybrid sites with improved size control, previously unknown composition and enhanced performance characteristics.

Summary of The Invention

The present disclosure provides nanoparticles (e.g., core or core-shell nanoparticles). The present disclosure also provides methods of making and using the nanoparticles.

In one aspect, the present disclosure provides a method of making functionalized nanoparticles (e.g., ultra-small functionalized nanoparticles). The methods may include methods of modifying the Pegylation Posterior Surface (PPSMI) by insertion, such as the (PPSMI) methods described herein.

The method is based on the reaction of nanoparticles (e.g., having a dimension such as the longest dimension) of 2 to 15nm (e.g., 2 to 10nm) that can be ultra-small nanoparticles of less than 10nm in size (e.g., 2 to 9.99nm in size) comprising a plurality of polyethylene glycol (PEG) groups, some or all of which can be functionalized with one or more functional groups or groups capable of reacting to form functional groups that are covalently bound to the surface of the nanoparticles (which can be referred to as pegylated nanoparticles), with one or more functionalized precursors comprising at least one reactive group. The resulting nanoparticles having one or more reactive groups covalently bound to the surface of the nanoparticles are then reacted with a functional group precursor capable of reacting with the reactive groups to produce nanoparticles having one or more functional groups covalently bound to the surface of the nanoparticles.

In one aspect, the present disclosure provides a composition comprising a nanoparticle of the present disclosure. The composition may comprise one or more types (e.g., having different average sizes and/or one or more different compositional characteristics). The composition can include functionalized nanoparticles having from 1 to 5 (e.g., 1, 2, 3, 4, or 5) types of different functional ligands located on (e.g., covalently bound to) the surface of the NP.

In one aspect, the present disclosure provides uses of the nanoparticles and compositions of the present disclosure. The ligand (functional group) carried by the nanoparticle may include a diagnostic and/or therapeutic agent (e.g., a drug). Thus, the nanoparticles or compositions comprising the nanoparticles are useful for delivery (e.g., therapeutic methods) and/or imaging methods.

Brief Description of Drawings

For a fuller understanding of the nature and objects of the present disclosure, reference should be made to the following detailed description taken together with the accompanying figures.

FIG. 1 shows a schematic of a C' point synthesis system incorporating a method for modifying the PEGylated rear surface (PPSMI) by insertion. Surface modification of the C' site with additional functional ligands can be achieved by either: (i) co-condensing different heterobifunctional PEG-silanes in a pegylation step followed by covalent attachment of functional ligands to the heterobifunctional PEG after pegylation, or (ii) covalently inserting silanes with functional groups (e.g., amines or thiols) between the PEG chains and on the silica surface in a post-pegylation step (PPSMI). The alternate pathways in each reaction step are shown using dashed lines. The particular choice of reaction route for the different multifunctional C' sites depends on the application requirements. The thiol-ene reaction for converting the SH-drug-C (RGDyC) -PEG-C 'site to the DFO-drug-C (RGDyC) -PEG-C' site is shown with a break in the black solid line (lower right panel). The inset in the upper right shows the molecular diagram of the pentafunctional C' site, with the synthetic pathway highlighted in light blue. These five functions include fluorescence by NIR fluorescent Cy5 dye inside the silica core, targeting cancer cells by specific c (rgdyc) peptides, small therapeutic EFV drugs attached to some PEG chains, radioisotope labeling by specific DFO chelators, and pH sensing by attachment of a second sensing molecule, FITC dye, between PEG chains. The silicon, oxygen, carbon, nitrogen, sulfur, chlorine and fluorine atoms are purple, red, gray, blue, yellow, green and aqua, respectively. For convenience of observation, no hydrogen atom is shown.

FIG. 2 shows the C (RGDyC) -PEG-Cy5.5-C' points of increased C (RGDyC) peptide number per NP. (A to C) GPC elution profiles of C (RGDyC) -PEG-Cy5.5-C 'spots synthesized using a standard C' spot synthesis protocol (A), or synthesized under conditions where the concentration of C (RGDyC) -PEG-silane is increased (B), or synthesized under conditions where the concentrations of C (RGDyC) -PEG-silane and ammonium hydroxide are simultaneously increased (C). (D and E) GPC elution Profile (D) and FCS correlation Curve (E) of purified C (RGDYC) -PEG-Cy5.5-C' dots containing an average of 58C (RGDYC) peptides/particle. (F) Comparison of UV-vis absorption spectra at the C (RGDyC) -PEG-Cy5.5-C' point with varying amounts of cRGDY ligand per NP, the amount of cRGDY ligand per NP is shown in the inset.

FIG. 3 shows the characterization of the C (RGDyC) -PEG-C' point functionalized with amine groups during (left) or after (right) PEGylation. (A and B) c (RGDyC) -PEG-NH before (A) and after (B) four rounds of GPC purification ₂ GPC elution pattern at Cy 5-C', amine functionalization being carried out in the PEGylation step. (C) Purified c (RGDyC) -PEG-NH ₂ FCS correlation curves and fits at Cy 5-C' point. (D) Comparison of UV-vis spectra of C (RGDyC) -PEG-Cy 5-C' spots with (red) and without (black) amine functionalization during PEGylation. (E and F) NH before (E) and after (F) purification ₂ -cRGDY-PEG-Cy 5-C' spot GPC elution profile with amine functionalization after PEGylation. (G) Purified NH ₂ FCS correlation curve and fitting of cRGDY-PEG-Cy 5-C' point. (H) Comparison of UV-vis spectra of C (rgdyc) -PEG-Cy 5-C' spots with (red) and without (black) amine functionalization after the pegylation step.

FIG. 4 shows PPSMI-based surface functionalization of the C (RGDyC) -PEG-C' site with a radioisotope chelator. (A and B) GPC elution profiles of DFO-C (RGDyC) -PEG-Cy5-C 'dots before (A) and after (B) purification by further linking DFO-NCS to derivatized amine-functionalized NH after PEGylation during one-pot NP synthesis for the DFO-C (RGDyC) -PEG-Cy 5-C' dots ₂ -cRGDY-PEG-Cy 5-C' spot to introduce DFO ligands. (C) FCS at purified DFO-C (RGDyC) -PEG-Cy 5-C' siteCorrelation curves and fitting. (D) Comparison of UV-vis spectra of spots of purified DFO-C (RGDyC) -PEG-Cy5-C synthesized using two different DFO-NCS concentrations. (E and F) GPC elution profiles of DOTA-C (RGDyC) -PEG-Cy5-C 'dots before (E) and after (F) purification by further linking DOTA-NCS to derivatized amine-functionalized NH after PEGylation during one-pot NP synthesis for the DOTA-C (RGDyC) -PEG-Cy 5-C' dots ₂ -C (rgdyc) -PEG-Cy 5-C' spot to introduce DOTA ligand. (G) FCS correlation curves and fits of purified DOTA-C (RGDyC) -PEG-Cy 5-C' spots. (H) Comparison of UV-vis spectra of spots of purified DOTA-C (RGDyC) -PEG-Cy 5-C' synthesized using two different concentrations of DOTA-NCS. The chemical structures of the DFO-NCS and DOTA-NCS chelators are shown in the insets of (A) and (E), respectively.

FIG. 5 shows a tetrafunctional C' site containing three types of functional ligands on the NP surface. (A) Comparison of points C' with stepwise increasing functionality as shown in the inset. (B and C) GPC elution Profile of purified FITC-DFO-C (RGDyC) -PEG-C' Point (B) and FCS correlation Curve with fitting (C). (D and E) emission spectra of purified FITC-DFO-C (RGDyC) -PEG-C' dots with excitation wavelengths of 500nm (D) and 650nm (E), respectively. (F) A ratio calibration curve was generated by dividing the peak sensor emission intensity (525nm) by the peak reference emission intensity (660nm) and plotting the pH. (A) The UV-vis absorption spectra shown in (1) were measured for the desired FITC signal in PBS buffer to estimate the number of FITC dyes per NP.

FIG. 6 shows a pentafunctional C' site containing four types of functional ligands on the NP surface. (A to C) GPC elution profile of pentafunctional FITC-DFO-EFV-C (RGDyC) -PEG-Cy 5-C' points (A), FCS correlation curve (B) and UV-vis spectra and fits (C). The chemical structure of the EFV drug is in (a). (D to H) deconvolution of UV-vis spectra (C) to contributions from PEG-Cy 5-C' dots (D), C (RGDyC) ((E)), EFV (F), DFO (G), and FITC (H). The deconvolution (deconvolution) was obtained by fitting the UV-vis spectrum (C) using a set of standard spectra (fig. 12B to D, fig. 15E and fig. 17C) consisting of each individual component.

FIG. 7 shows the nomenclature of the multifunctional C' points. Taking the site of trifunctional DFO-C (RGDyC) -PEG-Cy 5-C' as an example, the particle structure diagram is inserted in the lower left corner.

Figure 8 shows the C (rgdyc) -PEG-C' points of cancer targeting functionalized with different NIR dyes. (A and B) GPC elution profiles before (A) and after (B) purification of C (RGDyC) -PEG-Cy5.5-C' spots encapsulating Cy5.5 dye. (C) FCS correlation curve and fitting of purified C (RGDyC) -PEG-Cy5.5-C' point. (D) Comparison of UV-vis absorbance with and without the C (RGDyC) -functionalized PEG-Cy5.5-C' point. (E and F) GPC elution profiles before (E) and after (F) NP purified with C (RGDyC) -PEG-Cy 5-C' spots encapsulating Cy5 dye. (G) FCS correlation curve and fitting of purified C (RGDyC) -PEG-Cy 5-C' points. (H) Comparison of UV-vis absorbance at the point of PEG-Cy 5-C' with and without C (RGDyC) functionalization. (I and J) GPC elution profiles before (I) and after (J) NP purification of C (RGDyC) -PEG-CW 800-C' dots encapsulating CW800 dye. (K) FCS correlation curve and fitting of the purified cRGDY-PEG-CW 800-C' point. (L) comparison of UV-vis absorbance at 800-C' point with and without C (RGDyC) functionalized PEG-CW. The different absorption peaks at about 655nm (D), 657nm (H), and 795nm (L) indicate successful encapsulation of different types of NIR dyes.

FIG. 9 shows the characterization of the multifunctional C (RGDyC) -mal-PEG-Cy 5-C' dot, which was made by introducing mal-PEG-silane during PEGylation of NP. (A and B) GPC elution patterns before (A) and after (B) purification. Although typical C' spot purification is performed by two GPC cycles, for the C (RGDyC) -mal-PEG-Cy5-C spot, a total of four GPC cycles are required to obtain the desired product purity (as shown in (B)). (C) Representative FCS correlation curves and fits of purified C (RGDyC) -mal-PEG-Cy 5-C' spots. (D) Comparison of UV-vis absorption spectra of purified C (RGDyC) -mal-PEG-Cy 5-C' spots synthesized using different mal-PEG-silane concentrations. (D) The inset in (a) shows absorption at shorter wavelengths, which increases with increasing concentration of mal-PEG-silane, which can be attributed to increased maleimide group loading of the NP.

FIG. 10 shows the characterization of the multifunctional C (RGDyC) -DBCO-PEG-Cy5-C ' and C (RGDyC) -DFO-PEG-Cy5-C ' spots, which were prepared by introducing DFO-silane and DBCO-PEG-silane during PEGylation of NP, and C (RGDyC) -DFO-PEG-Cy5-C ' spots. (A and B) GPC elution profiles before (A) and after (B) purifying the C (RGDyC) -DBCO-PEG-Cy 5-C' spot synthesized by co-condensing DBCO-PEG-silane with C (RGDyC) -PEG-silane and PEG-silane during PEGylation. (C) Representative FCS correlation curves and fits of purified C (RGDyC) -DBCO-PEG-Cy 5-C' spots. (D) Comparison of UV-vis absorption spectra at the points of purified C (RGDyC) -DBCO-PEG-Cy 5-C' synthesized using different DBCO-PEG-silane concentrations. (E and F) GPC elution profiles before (E) and after (F) purifying the C (RGDyC) -DFO-PEG-Cy 5-C' spot synthesized by co-condensing DFO-silane with C (RGDyC) -PEG-silane and PEG-silane during PEGylation. (G) Representative FCS correlation curves and fits of purified C (RGDyC) -DFO-PEG-Cy 5-C' spots. (H) Comparison of UV-vis absorption spectra of purified C (RGDyC) -DFO-PEG-Cy 5-C' spots synthesized using different DFO-silane concentrations. The chemical structures of DBCO-silane and DFO-PEG-silane are shown as insets in (A) and (E), respectively. Although monodisperse sites C (RGDyC) -DBCO-PEG-Cy5-C 'and C (RGDyC) -DFO-PEG-Cy 5-C' can be generated by this method, the resulting NPs exhibit poor conjugation activity due to limited ligand accessibility and unwanted ligand distribution.

FIG. 11 shows a comparison of UV-vis absorption spectra of the point C (RGDyC) -PEG-Cy 5-C' functionalized by DFO using various methods. When the C (RGDyC) -PEG-Cy5-C 'spot was mixed with the DFO-silane conjugate, the absorbance spectrum of the resulting NP was not substantially different, indicating lower reaction efficiency, compared to the unfunctionalized C (RGDyC) -PEG-Cy 5-C' spot. In addition, the cRGDY peptide carries a primary amine group, which can potentially react with DFO-NCS. The absorption spectrum of the product of the synthesis reaction between the (rgdyc) -PEG-Cy 5-C' site and DFO-NCS also did not show any other absorption features around 245nm, indicating that the conversion yield of this DFO conjugation was negligible (the results also indicate that the primary amine group on the C (rgdyc) peptide has limited reactivity with DFO-NCS). The average number of DFO groups per C' point in these experiments was below 0.5. In contrast, when DFO-NCS is reacted with amine-functionalized NH derived from the surface modification reaction with amino-silane after PEGylation ₂ The most preferred is the mixing of the (C) (RGDyC) -PEG-Cy 5-C' spotsThe final C 'point product will show significant absorbance below 300nm, indicating that there is significant DFO attachment, the amount of which by calibration can reach an average of-20 molecules of DFO per C' point. These data indicate that the DFO group at the DFO-C (RGDyC) -PEG-Cy 5-C' site is attached primarily through the amine group introduced after PEGylation, rather than to the primary amine group on the C (RGDyC) peptide.

Figure 12 shows the estimation of the number of ligands per multifunctional C' dot containing more than one type of functional ligand on the surface by deconvolution absorption spectra. (A) Comparison of UV-vis absorption spectra of C 'spots with stepwise increasing functionality, the absorption spectra (B to D) of each component of the DFO-C (RGDyC) -PEG-Cy 5-C' spots were generated on the basis thereof. The absorption signal of the C (RGDyC) peptide on the surface of the C ' spot (green curve in C) was obtained by subtracting the absorption signal of the PEG-Cy5-C ' spot (red curve in A/B) from the absorption signal of the C (RGDyC) -PEG-Cy5-C ' spot (green curve in A). The absorption signal of the DFO chelator on the surface of the C ' spot (blue curve in D) was obtained by subtracting the absorption signal at the site cRGDY-PEG-Cy5-C ' (green curve in A) from the absorption signal at the site DFO-C (RGDyC) -PEG-Cy5-C ' (blue curve in A). The resulting spectra for C (rgdyc) and DFO were then compared with the absorbance signals of free C (rgdyc) peptide (top green curve in C) and DFO-silane molecule (top blue curve in D), respectively (C and D), to confirm the identity of the spectra. The use of conjugated DFO-silanes instead of DFO-NCS molecules for comparison with DFO on NPs was because conjugation of DFO-NCS to amine functionalized ligands (e.g., amino-silanes) resulted in a large change in the absorption spectrum (data not shown). Thus, the absorption of DFO-silane is more consistent with the DFO group at the C' point than with free DFO-NCS. The resulting PEG-Cy5-C 'spot (B), C (RGDyC) at the C' spot (lower green curve in C), and DFO (lower blue curve in D) at the C 'spot were normalized and used as standards to fit absorption spectra (E) of samples from other DFO-C (RGDyC) -PEG-Cy 5-C' spots. The fitting equation is a linear combination of the absorption intensities of the different components, i.e. F (A, B, C, D) ═ A × I _{PEG-Cy 5-C' dot} +B*I _c(RGDyC) +C*I _DFO + D, which describes the sample wellTanspect spectrum (E). Parameter D is added to the fit equation to correct for possible baseline shifts of the UV-vis setting. Fitting deconvoluted sample spectra (E) to the contribution of the individual components (F to H), the number of Cy5, c (rgdyc) and DFO molecules per NP was estimated to be about 1.6, 23 and 4, respectively, from the extinction coefficients therein by using Cy5 free dye, free c (rgdyc) peptide and free DFO-silane molecules. Although the extinction coefficient of Cy5 was used according to the manufacturer's report, the results of the insertions in (C) and (D) were used to separately calibrate the extinction coefficients of free C (rgdyc) peptide and free DFO-silane molecules, respectively.

Fig. 13 shows the generalization of surface modification after pegylation by using other types of conjugation chemistry. (A to C) GPC elution profile (A), FCS correlation curve and fitting (B) and UV-vis absorption spectra (C) of purified thiol-functionalized SH-PEG-Cy5-C 'spots synthesized by introducing thiol-silane into the reaction mixture at PEG-Cy 5-C' spot after NP PEGylation but before purification. A slight increase in absorption below 250nm was observed compared to NPs without additional thiol functionalization (C). (D to F) GPC elution Profile (D), FCS correlation Curve and fitting (E) and UV-vis absorption Spectroscopy (F) of purified FITC-PEG-Cy5-C 'dots synthesized by further incorporating FITC-NCS dye into the reaction mixture of thiol-functionalized SH-PEG-Cy 5-C' dots after addition of thiol-silane but before NP purification. The absorption signal at the FITC-PEG-Cy5-C 'spot was significantly increased at a wavelength of about 450nm (F) compared to the product from the same synthesis using PEG-Cy 5-C' spot without thiol functionalization. This signal corresponds to the absorption signal of the FITC dye in DI water, confirming the successful attachment of FITC and the accessibility of the thiol group at the SH-PEG-Cy 5-C' site. Note that lower FITC signal was also observed in samples without thiol functionalization, probably due to non-specific adsorption of FITC dye to the C 'spot or due to residual thiol groups generated in the standard C' spot synthesis due to excess thiol-silane used in the Cy5 dye silane conjugation step.

FIG. 14 shows amine functionalized multifunctional C' dotsStability of (2). Purified NH at 6 month time point after NP manufacture ₂ GPC elution patterns of (A) C (RGDyC) -PEG-Cy5-C 'spot and (B) DFO-C (RGDyC) -PEG-Cy 5-C' spot. In both cases, the NPs were stored in PBS buffer at 4 ℃ during the stability test. NH (NH) ₂ The elution profile (a) of the (C) (rgdyc) -PEG-Cy 5-C' spot shows smaller additional peaks, which correlate with smaller molar mass products, suggesting that NP degradation may be caused by primary amine groups on the silica surface. In contrast, DFO-C (RGDyC) -PEG-Cy 5-C' spot (B) remained stable throughout the stability test.

FIG. 15 shows a deconvolution of the absorption spectra for the multifunctional C' spot containing three types of functional ligands on the NP surface. (A) UV-vis absorption spectra of FITC-DFO-C (RGDyC) -PEG-Cy5-C 'spot, which can be fit well by using a linear combination of the absorption signals of PEG-Cy 5-C' spot, C (RGDyC), DFO and FITC. This deconvolution is similar to that described for DFO-C (RGDyC) -PEG-Cy 5-C' spot in FIG. 12, but also includes the UV-vis signal of FITC in the analysis. Spectra (A) were deconvoluted to contributions from PEG-Cy 5-C' point (B), C (RGDyC) ((C)), DFO (D), and FITC (E), according to the fit. From these contributions of the fit, the number of Cy5, c (rgdyc), DFO and FITC molecules was estimated to be about 1.7, 23, 19 and 4, respectively, for each NP.

Figure 16 shows the characterization of tetrafunctional C' points obtained by orthogonal surface functionalization and deconvolution synthesis of the absorption spectra. (A to C) GPC elution profile from deconvolution of tetrafunctional FITC-DFO-C (RGDyC) -PEG-Cy 5-C' points (A), FCS correlation curve and fitting (B), and UV-vis spectroscopy (C) and fitting, where FITC and DFO groups were introduced simultaneously, rather than step-wise attached (compare results in FIG. 15). (D to G) deconvolution of the UV-vis spectra (C) to contributions from PEG-Cy 5-C' dots (D), C (RGDyC) (E), DFO (F) and FITC (G). From these contributions in the fit, the number of Cy5, c (rgdyc), DFO and FITC molecules was estimated to be about 1.9, 22, 4 and 3, respectively, for each NP.

Figure 17 shows the attachment of small therapeutic drugs for the preparation of five-functional C-sites. (A) Conjugation reaction of EFV and azido-PEG-thiol to functionalize the EFV drug molecule with thiol groups. The resulting EFV-PEG-thiol molecule was then attached to the C (rgdyc) -mal-PEG-Cy 5-C' site by thiol-ene reaction after NP pegylation and before introduction of DFO and FITC. (B) UV-vis spectra of FITC-DFO-EFV-C (RGDyC) -PEG-Cy5-C 'spot and FITC-DFO-C (RGDyC) -mal-PEG-Cy 5-C' spot. These two samples were obtained from the same reaction batch with or without addition of EFV-PEG-thiol. (C) Standard absorption spectrum of EFV at the C ' point obtained by subtracting the absorption value at the FITC-DFO-C (RGDyC) -mal-PEG-Cy5-C ' point from the absorption value at the FITC-DFO-EFV-C (RGDyC) -PEG-Cy5-C ' point. The resulting spectrum is consistent with the UV-vis spectrum of the free EFV drug, indicating successful attachment of the EFV to the C' site. The calibration of the extinction coefficient of free EFV drug molecules is inserted in the (C) diagram.

FIG. 18 shows the characterization of the DBCO-PEG-Cy 5-C' spot. (A) Comparison of UV-vis absorption spectra and deconvolution fit of DBCO-PEG-Cy 5-C' spots with different DBCO ligand numbers. (B to G) UV-vis absorbance deconvolution of DBCO-PEG-Cy 5-C' points with 32(B, D and F) and 64(C, E and G) DBCO groups per particle (B and C), GPC elution profile and fit (D and E), and FCS correlation curve and fit (G and G), respectively.

FIG. 19 shows the characterization of DBCO-DFO-PEG-Cy 5-C' spot. (A) UV-vis absorption spectra and deconvolution fitting. (B) UV-vis absorbance deconvolution. (C) GPC elution profile and fit. (D) FCS correlation curve and fitting.

Detailed Description

The present disclosure provides nanoparticles (e.g., core or core-shell nanoparticles). In various examples, the nanoparticles are ultra-small nanoparticles. The present disclosure also provides methods of making and using the nanoparticles.

Unless otherwise indicated, all ranges provided herein include all values falling within the tenth decimal place range.

As used herein, unless otherwise specified, the term "group," when used in the context of a chemical structure, refers to a chemical entity having one or more ends that can be covalently bonded to other chemical species. Non-limiting illustrative examples of groups include:

other non-limiting illustrative examples of groups include:

the term "alkyl" as used herein, unless otherwise indicated, refers to a branched or unbranched saturated hydrocarbon group. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, isopropyl, tert-butyl, and the like. For example, the alkyl group may be C ₁ To C ₈ Alkyl, which includes all integers and ranges of carbon numbers therebetween (e.g., C) ₁ 、C ₂ 、C ₃ 、C ₄ 、C ₅ 、C ₆ 、C ₇ And C ₈ ). An alkyl group may be unsubstituted or substituted with one or more substituents. Examples of substituents include, but are not limited to, various substituents, such as halogens (-F, -Cl, -Br, and-I), aliphatic groups (e.g., alkyl, alkenyl, alkynyl), aryl groups, alkoxy groups, amine groups, carboxylate groups, carboxylic acids, ether groups, alcohol groups, alkynyl groups (e.g., ethynyl), and the like, and combinations thereof.

The technology disclosed herein provides a method for aqueous phase synthesis of ultra-small functional pegylated fluorescent silica nanoparticles that improves control in a number of aspects including particle size, particle size distribution, fluorescence wavelength, fluorescence brightness, composition, particle pegylation, particle surface functionalization, synthesis yield, product purity, and manufacturing reliability. Precise control of the systematics covering these aspects in a single organic-inorganic hybrid nanomaterial synthesis system has never been achieved before, preventing the safe conversion of organic-inorganic hybrid nanomaterials from the laboratory to the clinic. Thus, the technology disclosed herein provides a way to obtain well-defined and adjustable at the system level silica-based nanomaterials that show great potential in nanomedical applications.

Disclosed herein is a general surface modification method to, for example, modularly and orthogonally functionalize nanoparticles (e.g., Cornell prime dots (C' dots), which may be, for example, nanoparticles described in this disclosure (e.g., ultra-small PEG-core or core-shell silica or aluminum silicon nanoparticles below 10nm) with one to five (e.g., 1, 2, 3, 4, or 5) different functional ligands on the NP surface). The surface modification methods utilize gaps in PEG groups located on the surface of the nanoparticles and anchoring groups that can be inserted by using, for example, amine and/or thiol functionalized silane molecules that can be functionalized with various functional ligands.

In one example, the modification method provides for the synthesis of pentafunctional nanoparticles (e.g., C' sites) that integrate multiple properties, e.g., fluorescence detection, specific cell targeting, radioisotope chelation/labeling, ratiometric pH sensing, drug delivery, or combinations thereof, into a single NP while maintaining the overall NP size at, e.g., less than 10nn (e.g., less than 8nm or less than 7 nm). This is achieved by exploiting the fact that the PEG layer at the C' site can be penetrated by small molecules.

For example, amine and/or thiol functionalized silane molecules can be inserted between PEG chains and on the silica surface of the nanoparticle (e.g., C' dots), and additional functional ligands can then be attached to the amine and/or thiol functionalized silane molecules. This method of modifying the pegylation back surface (PPSMI) by insertion requires only a few additional steps between pegylation and purification of nanoparticles (e.g., C' dots) in a one-pot water-based synthesis, while not reducing the production of high quality NPs. The resulting nanoparticles with additional functionality (e.g., C' dots) exhibit physicochemical properties such as their size and PEG density close to that of clinically transformed nanoparticles (e.g., C dots), opening the door for diversification of their clinical applications. Modification of nanoparticle synthesis (e.g., C' site synthesis) allows, for example, a large number of targeting peptides per particle, and a simple and versatile spectroscopic method to quantitatively assess specific numbers of different surface ligands by deconvoluting the absorption spectra into individual components.

In one aspect, the present disclosure provides a method of making functionalized nanoparticles (e.g., ultra-small functionalized nanoparticles). The process may include a PPSM) process, for example, the PPSMI process described herein. The process is based on the use of an aqueous reaction medium, such as water. In one example, one or more nanoparticles described in the present disclosure (e.g., nanoparticles of a composition) are made by the methods described in the present disclosure.

The method is based on the reaction of nanoparticles (e.g., having a dimension such as the longest dimension) of 2 to 15nm (e.g., 2 to 10nm) which may be ultra-small nanoparticles of less than 10nm in size (e.g., 2 to 9.99nm in size) comprising a plurality of polyethylene glycol (PEG) groups, some or all of which may be functionalized with one or more functional groups or groups capable of reacting to form functional groups, with one or more functionalized precursors comprising at least one reactive group, the PEG groups being covalently bound to the surface of the nanoparticle (which may be referred to as pegylated nanoparticles). Reacting the resulting nanoparticle having one or more reactive groups covalently bound to the surface of the nanoparticle with a functional group precursor capable of reacting with the reactive group, thereby producing a nanoparticle having one or more functional groups covalently bound to the surface of the nanoparticle.

The process may be a "one-pot" reaction. The method may also include a separate reaction. The separate reactions can have reaction mixtures (e.g., nanoparticles, functionalized precursors, functional group precursors, solvents, and the like, as well as various combinations thereof) with the same or different reactants. The nanoparticles may be separated between any individual reactions.

In one example, the pegylated nanoparticles are reacted with one or more functionalized precursors and one or more functional group precursors. The reactions may be performed in any order, as long as the nanoparticles are first reacted with at least one functionalizing precursor. For example, nanoparticles having a single type of reactive group are reacted with one or more functional group precursors. In another example, nanoparticles having two or more structurally and/or chemically distinct reactive groups (e.g., 2, 3, 4, or 5 structurally and/or chemically distinct reactive groups) are reacted with two or more distinct functional group precursors (e.g., 2, 3, 4, or 5 structurally and/or chemically distinct functional group precursors), where the individual reactive groups/functional group precursors may have orthogonal reactivity.

Various conjugation chemistries/reactions can be used to covalently attach functional groups to the surface of the nanoparticles. Thus, the functionalized precursor may comprise various reactive groups. Many suitable conjugation chemistries and reactions are known in the art. In various examples, the reactive group is a reactive group known in the art to react in a particular conjugation chemistry or reaction, and the functional group precursor comprises a complementary group of the particular conjugation chemistry/reaction known in the art.

The functionalized precursor comprises one or more reactive groups and groups (e.g., silane groups) that can react with the surface of the nanoparticle to form covalent bonds. The reactive group may react with the functional group precursor to form a functional group that is covalently bound to the surface of the nanoparticle. Non-limiting examples of reactive groups include amine groups, thiol groups, carboxylic acid groups, carboxylate groups, ester groups (e.g., activated ester groups), maleimide groups, allyl groups, terminal alkyne groups, azide groups, thiocyanate groups, and combinations thereof. Examples of functionalized precursors are known in the art and are commercially available or can be prepared using methods known in the art.

In various examples, the functionalized precursor comprises a silane group comprising one or more-Si-OH groups (e.g., 1, 2, or 3 Si-OH groups) and at least one reactive group (e.g., 1, 2, or 3 reactive groups). The silane group and reactive group can be covalently linked through a linking group, for example, an alkyl group (e.g., C) ₁ 、C ₂ 、C ₃ 、C ₄ 、C ₅ 、C ₆ 、C ₇ Or C ₈ Alkyl groups). Without being bound by any particular theory, it is believed that the Si-OH groups of the functionalized precursor can interact with the nano-scaleThe surface hydroxyl groups (e.g., surface Si-OH groups) of the particles react.

A nanoparticle or nanoparticles can be reacted to form various numbers of reactive and/or functional groups. For example, reacting the nanoparticle or nanoparticles to form 1 to 100 reactive groups and/or functional groups, including all integers and ranges therebetween (e.g., reacting the nanoparticles such that for each nanoparticle of the plurality of nanoparticles an average of 1 to 100 groups and/or functional groups are formed, including all integers and ranges therebetween), covalently bonds to the surface of the nanoparticle or nanoparticles. In various examples, it is within the ability of one skilled in the art to react a nanoparticle or nanoparticles to form 20 to 100, 25 to 100, 30 to 100, 35 to 100, 40 to 100, or 50 to 100 groups and/or functional groups (e.g., a plurality of nanoparticles can be reacted to form an average of 20 to 100, 25 to 100, 30 to 100, 35 to 100, 40 to 100, or 50 to 100 groups and/or functional groups that are covalently bound to the surface of each of the nanoparticles).

The functional group precursor may react with a reactive group of the nanoparticle to form a functional group covalently bound to the surface of the nanoparticle. The functional group precursor comprises a functional group (e.g., a dye group, a chelating group, a targeting group, a drug group, a radiolabel/isotope group, etc., which may be derived from a dye molecule, a chelator molecule, a targeting molecule, etc.) and a group capable of reacting with a reactive group of a nanoparticle. Non-limiting examples of groups that react with a reactive group include amine groups, thiol groups, carboxylic acid groups, carboxylate groups, ester groups (e.g., activated ester groups), maleimide groups, allyl groups, terminal alkyne groups, azide groups, thiocyanate groups, and combinations thereof. In various examples, the functional group precursor comprises one or more groups known in the art to react in a particular conjugation chemistry or reaction (e.g., the functional group precursor comprises one or more groups known in the art to be complementary to a reactive group (e.g., a terminal alkynyl group) of the nanoparticle in a particular conjugation chemistry/reaction (e.g., click chemistry)). Examples of functional group precursors are known in the art and are commercially available or can be prepared using methods known in the art.

Various functional groups are known in the art. The functional group is also referred to herein as a ligand. Functional groups can have a variety of functionalities (e.g., absorption/emission behaviors such as fluorescence and phosphorescence, which can be used for imaging, sensing functionalities (e.g., pH sensing, ion sensing, oxygen sensing, biomolecule sensing, temperature sensing, etc.), chelating capabilities, targeting capabilities (e.g., antibody fragments, aptamers, proteins/peptides (natural, truncated, or synthetic), nucleic acids (such as DNA and RNA, etc.)), diagnostic capabilities (e.g., radioisotopes), therapeutic capabilities (e.g., drugs, nucleic acids, etc.), and the like, as well as combinations thereof.

The functional groups carried by the nanoparticles may include diagnostic and/or therapeutic agents (e.g., radioisotopes, drugs, nucleic acids, etc.). The nanoparticles may comprise a combination of different functional groups.

Non-limiting examples of therapeutic agents (which may be drugs) include, but are not limited to, chemotherapeutic agents, antibiotics, antifungal agents, antiparasitic agents, antiviral agents, and combinations thereof, as well as derivatives thereof. Examples of suitable drugs/agents are known in the art.

The nanoparticles may comprise various dyes (e.g., functional groups formed from various dyes). In various examples, the dye is an organic dye. In one example, the dye does not contain a metal atom. Non-limiting examples of dyes include fluorescent dyes (e.g., Near Infrared (NIR) dyes), phosphorescent dyes, non-fluorescent dyes (e.g., non-fluorescent dyes that exhibit fluorescence quantum yields of less than 1%), fluorescent proteins (e.g., EBFP2 (variant of blue fluorescent protein), mCFP (cyan fluorescent protein), GFP (green fluorescent protein), mCherry (variant of red fluorescent protein), iRFP720 (near infrared fluorescent protein)), and the like, and derivatives thereof. In various examples, the dye absorbs in the UV visible portion of the electromagnetic spectrum. In various examples, the dye has excitation and/or emission in the near infrared portion of the electromagnetic spectrum (e.g., 650-900 nm).

Non-limiting examples of organic dyes include cyanine dyes (e.g.,etc.), carbapenem (carborhodomine) dye (e.g., ATTO647N (available from ATTO-TEC and Sigma)Available), BODIPY dyes (e.g., BODIPY 650/665, etc.), xanthene dyes (e.g., fluorescein dyes such as Fluorescein Isothiocyanate (FITC), rose bengal, etc.), eosins (e.g., eosin Y, etc.) and rhodamines (e.g., TAMRA, Tetramethylrhodamine (TMR), TRITC, etc.),633. Alexa 633, HiLyte 594, etc.),DY800、DY782 and800CW, and the like, as well as derivative groups thereof.

The nanoparticles may comprise various sensing groups. Non-limiting examples of sensing groups include pH sensing groups, ion sensing groups, oxygen sensing groups, biomolecule sensing groups, temperature sensing groups, and the like. Examples of suitable sensing compounds/groups are known in the art.

The nanoparticles may comprise various chelating groups. Non-limiting examples of chelating groups include Desferrioxamine (DFO), 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid (DOTA), 1,4, 7-triazacyclononane-1, 4, 7-triacetic acid (NOTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrins, and the like, as well as derivatives thereof. The chelating group may comprise a radioisotope. Examples of radioisotopes are described herein and known in the art.

The radioisotope can be a functional group. The radioisotope may be a diagnostic and/or therapeutic agent. For example, by introducing radioactive isotopes, e.g. ¹²⁴ I for Positron Emission Tomography (PET). Non-limiting examples of radioisotopes include ¹²⁴ I、 ¹³¹ I、 ²²⁵ Ac、 ¹⁷⁷ Lu, and the like. The radioisotope can be chelated to a chelating group.

Targeting groups may also be conjugated to the nanoparticles to allow targeted delivery of the nanoparticle or nanoparticles. The targeting group may be formed by (derived from) a targeting molecule. For example, targeting groups capable of binding to cellular components associated with a particular cell type (e.g., on the cell membrane or in the intracellular compartment) are conjugated to the nanoparticles. The targeting group may be a tumor marker or a molecule in the signaling pathway. The targeting group may have specific binding affinity for certain cell types (e.g., tumor cells). In certain examples, targeting groups can be used to direct the nanoparticles to specific regions, such as the liver, spleen, brain, and the like. Imaging can be used to determine the location of the nanoparticles in the individual. Examples of targeting groups include, but are not limited to, linear and cyclic peptides (e.g., targeting alpha) _v β ₃ Cyclic (arginine-glycine-aspartic acid, tyrosine-cysteine) peptides of integrins, c (rgdyc), etc.), antibody fragments, various DNA and RNA fragments (e.g., siRNA).

As used herein, unless otherwise specified, the term "derivatize" refers to the formation of a group by reaction of a native functional group of a compound (e.g., a group formed by reaction of an amine of a compound with a carboxylic acid) or the chemical modification of a compound to introduce a new chemically reactive group on the compound that reacts to form a group.

The methods described herein can be scaled up linearly, for example, from 10ml reaction to 1000ml or more, without any substantial change in product quality. This scalability is important for large-scale production of nanoparticles.

The process may be carried out in an aqueous reaction medium (e.g., water). For example, the aqueous medium comprises water. Certain reactants are added to the various reaction mixtures as solutions in polar aprotic solvents (e.g., DMSO or DMF). In various examples, the aqueous medium does not contain an organic solvent (e.g., an alcohol, such as C) other than 10% or more, 20% or more, or 30% or more of the polar aprotic solvent ₁ To C ₆ Alcohol). In one example, the aqueous medium does not contain 1% or more, 2% or more, 3% or more, 4% or more, or 5% or more alcohol. In one example, the aqueous medium does not contain any detectable alcohol. For example, the reaction medium of any step of any of the methods disclosed herein consists essentially of water and, optionally, a polar aprotic solvent.

At various points in the process, the pH may be adjusted to a desired value or within a desired range. The pH of the reaction mixture can be raised by the addition of a base. Non-limiting examples of suitable bases include ammonium hydroxide.

Various nanoparticles may be used. Non-limiting examples of nanoparticles include silica nanoparticles and aluminosilicate nanoparticles. The nanoparticle may be a core-shell nanoparticle. The nanoparticles are surface functionalized (e.g., pegylated) with polyethylene glycol groups, some or all of which may be functionalized with one or more functional groups or groups capable of reacting to form functional groups. The nanoparticles may comprise PEG groups having groups capable of reacting to form functional groups. These nanoparticles may be functionalized with one or more functional groups. For example, a functionalized ligand (functional group precursor) is reacted with a reactive group of a PEG group. Examples of suitable reaction chemistries and conditions for functionalizing PEG groups after nanoparticle synthesis are known in the art. Suitable nanoparticles and methods of making such nanoparticles are disclosed in U.S. patent application No. 15/571,420, filed on 2017, 11/2, the disclosure of which is incorporated herein by reference.

For example, a method of preparing a nanoparticle or core-shell nanoparticle surface functionalized (i.e., pegylated) with polyethylene glycol groups, the method comprising: a) forming a reaction mixture at room temperature (e.g., 15 ℃ to 25 ℃, depending on location), the reaction mixture comprising water and TMOS (silica core-forming monomer) (e.g., at a concentration of 11mM to 270mM), wherein the pH of the reaction mixture (which can be adjusted using a base such as ammonium hydroxide) is 6 to 9 (which results in the formation of core precursor nanoparticles having an average size (e.g., longest dimension) of, for example, 1nm to 2 nm); b) i) maintaining the reaction mixture at a time (t) ¹ ) And temperature (T) ¹ ) (e.g. at room temperature to 95 deg.C (T) ¹ ) (t) of ¹ ) From 0.5 to 7 days) to form nanoparticles (core nanoparticles) having an average size (e.g. longest dimension) of from 2 to 15nm, or ii) if necessary, cooling the reaction mixture to room temperature and adding a shell-forming monomer (in addition to TMOS, for example tetraethylorthosilicate, such as TEOS or TPOS) to the reaction mixture from a) (said addition being carried out such that the concentration of shell-forming monomer is below the threshold for secondary nucleation) to form core-shell nanoparticles having an average size (e.g. longest dimension) of from 2 to 50nm (e.g. from 2 to 15 nm); c) if necessary, adjusting the pH of a reaction mixture comprising the core nanoparticles or the core-shell nanoparticles from b) i) or b) ii) to a pH of 6 to 10; and d) optionally, adding a PEG-silane conjugate (e.g., at a concentration of 10mM to 60mM) (e.g., a PEG-silane conjugate dissolved in a polar aprotic solvent such as DMSO or DMF) to the reaction mixture comprising the core nanoparticle or core-shell nanoparticle from b) i) or b) ii), respectively, at room temperature (e.g., to a reaction mixture comprising the core nanoparticle or core-shell nanoparticle covalently bound to a silane group (e.g., to thereby pegylate the core nanoparticle or core-shell nanoparticle), and maintaining the resulting reaction mixture for a time (t) at ² ) And temperature (T) ² ) Below (e.g. at room temperature (T) ² ) (t) of ² ) Is 0.5 minFrom one to 24 hours) (whereby at least a portion of the PEG-silane conjugate molecules are adsorbed onto at least a portion of the surface of the core nanoparticles or core-shell nanoparticles from b)); e) at time (t) ³ ) And temperature (T) ³ ) Heating the mixture from d) (e.g. at 40 ℃ to 100 ℃ (T) ³ ) (t) of ³ ) From 1 hour to 24 hours) to form a nanoparticle surface functionalized with polyethylene glycol groups or a core-shell nanoparticle surface functionalized with polyethylene glycol groups.

The nanoparticles may be subjected to post-synthesis processing steps. For example, after synthesis (e.g., after e in the above example), the solution is cooled to room temperature and then transferred to a dialysis membrane tube (e.g., a dialysis membrane tube with a molecular weight cut-off of 10,000, which is commercially available (e.g., from Pierce)). The solution in the dialysis tubing is dialyzed against DI water (water volume 200 times the reaction volume, e.g., 2000ml water for 10ml reaction) and the water is changed daily for 1 to6 days to wash away the remaining reagents, e.g., ammonium hydroxide and free silane molecules. The particles were then filtered through a 200nm syringe filter (brand fisher) to remove aggregates or dust. If necessary, additional purification methods including gel permeation chromatography and high performance liquid chromatography may be applied to the nanoparticles to further ensure high purification of the synthesized particles (e.g., with 1% or less unreacted reagents or aggregates). After any purification process, the purified nanoparticles can be transferred back into deionized water if other solvents are used in the alternative process.

The core may be a silicon core. The reaction mixture for silicon nucleus formation may contain TMOS as the only silicon nucleus-forming monomer.

The core may be an aluminosilicate core. The reaction mixture for aluminosilicate core formation may comprise TMOS as the only silicon core-forming monomer and one or more alumina core-forming monomers (e.g., aluminum alkoxides such as aluminum tri-sec-butoxide or a combination of aluminum alkoxides).

In the case of the synthesis of aluminosilicate cores, the pH of the reaction mixture is adjusted to a pH of 1 to 2 before the addition of the alumina core-forming monomers. After the aluminosilicate nuclei are formed, the pH of the solution is adjusted to a pH of 7 to 9, and optionally, PEG having a molecular weight between 100 and 1,000g/mol (including all integer values and ranges therebetween) is added to the reaction mixture at a concentration of 10mM to 75mM (including all integer mM values and ranges therebetween) before the pH of the reaction mixture is adjusted to a pH of 7 to 9.

The reaction mixture used to form the core nanoparticles may also include a dye precursor. In this case, the resulting core or core-shell nanoparticle has one or more dye molecules encapsulated or incorporated therein. For example, the core nanoparticle has 1, 2, 3, 4, 5, 6, or 7 dye molecules encapsulated therein. Mixtures of dye precursors may be used. The dye precursor is a dye conjugated to a silane. For example, dyes with maleimide functionality are conjugated to thiol-functionalized silanes. In another example, a dye with NHS ester functionality is conjugated to an amine-functionalized silane. Examples of suitable silanes and conjugation chemistry are known in the art. The dye may have an emission (e.g., fluorescence) wavelength of 400nm (blue) to 800nm (near infrared). For example, the dye is a NIR dye. Examples of suitable dyes are described herein. In various examples, the surface of a nanoparticle functionalized with polyethylene glycol groups or the surface of a core-shell nanoparticle functionalized with polyethylene glycol groups has one or more fluorescent dye molecules encapsulated therein.

A silica shell may be formed on the core nanoparticle. For example, the silica shell is formed after the core is formed. Examples of the precursor forming the silica shell include tetraalkyl orthosilicate such as TEOS and TPOS. Mixtures of precursors that form the silica shell may be used. TMOS is not a precursor for forming the silica shell. The silica shell forming precursor may be added to the reaction mixture as a solution in a polar aprotic solvent. Examples of suitable polar aprotic solvents include DMSO and DMF.

The precursor forming the silica shell needs to be added in separate aliquots. For example, the shell-forming monomers are added in separate aliquots (e.g., 40 to 500 aliquots). The aliquot may comprise one or more shell-forming precursors (e.g., TEOS and/or TPOS) and a polar aprotic solvent such as DMSO. Each aliquot may have 1 to 20 micromoles of shell-forming monomers. The interval between aliquot additions may be 1 to 60 minutes, including all integer minute values and ranges therebetween. The pH of the reaction mixture may vary during the formation of the silica shell. The pH needs to be adjusted to maintain a pH of 7-8.

After forming the core or core-shell nanoparticle, the core or core-shell nanoparticle may be reacted with one or more PEG-silane conjugates. The various PEG-silane conjugates can be added together or in various orders. This process is also referred to herein as pegylation. The percent conversion of PEG-silane is between 5% and 40%, and the polyethylene glycol surface density is 1.3 to 2.1 polyethylene glycol molecules/nm ² . The percent conversion of ligand-functionalized PEG-silane is 40% to 100% and the number of ligand-functionalized PEG-silane precursors reacted with each particle is 3 to 90.

Pegylation can be performed at different temperatures at different times. For example, for silica core or core-shell nanoparticles, pegylation can be performed by contacting the nanoparticles at room temperature for 0.5 minutes to 24 hours (e.g., overnight). For example, for alumina-silicate nanoparticles (e.g., alumina-silicate core nanoparticles or silica core silica shell nanoparticles), the temperature is 80 ℃ overnight.

The chain length of the PEG groups of the PEG-silane (i.e., the molecular weight of the PEG groups) can be adjusted to 3 to 24 ethylene glycol monomers (e.g., 3 to6, 3 to 9, 6 to 9, 8 to 12, or 8 to 24 ethylene glycol monomers). The PEG chain length of the PEG-silane can be selected to tailor the thickness of the PEG layer surrounding the particle and the pharmacokinetic profile of the pegylated particle. The PEG chain length of the ligand functionalized PEG-silane can be used to adjust the accessibility of the ligand groups on the PEG layer surface of the particle, thereby altering the binding and targeting properties.

The PEG-silane conjugate may comprise a ligand. The ligand is covalently bound to the PEG group of the PEG-silane conjugate (e.g., via the hydroxyl terminus of the PEG-silane conjugate). The ligand may be conjugated to the end of the PEG group opposite the end conjugated to the silane group. PEG-silane conjugates can be formed using heterobifunctional PEG compounds (e.g., maleimide functionalized heterobifunctional PEG, NHS ester functionalized heterobifunctional PEG, amine functionalized heterobifunctional PEG, thiol functionalized heterobifunctional PEG, and the like). Examples of suitable ligands (functional groups) are described herein.

For example, in addition to PEG-silane (e.g., as described in example d) above), a PEG-silane conjugate comprising a ligand is added. In this case, a nanoparticle surface functionalized with polyethylene glycol groups and polyethylene glycol groups containing ligands or a core-shell nanoparticle surface functionalized with polyethylene glycol groups and polyethylene glycol groups containing ligands is formed. The percent conversion of ligand-functionalized or reactive group-functionalized PEG-silane is from 40% to 100%, and the number of ligand-functionalized PEG-silane precursors reacted with each particle is from 3 to 600.

For example, a PEG-silane conjugate comprising a ligand (e.g., at a concentration of between 0.05mM and 2.5 mM) is added to a reaction mixture comprising a core nanoparticle or a core-shell nanoparticle (e.g., from example b) i) or b) ii), respectively, at room temperature before or after (e.g., 20 seconds to 5 minutes before or after) the addition of the PEG-silane conjugate (e.g., as described in example d) above). The resulting reaction mixture is maintained for a time (t) ⁴ ) And temperature (T) ⁴ ) Below (e.g. at room temperature (T) ⁴ ) (t) of ⁴ ) From 0.5 minutes to 24 hours) where at least a portion of the PEG-silane conjugated molecule is adsorbed on at least a portion of the surface of the core nanoparticle or core-shell nanoparticle (e.g., b) from the above example). Subsequently, at time (t) ⁵ ) And temperature (T) ⁵ ) The reaction mixture is heated (e.g. at 40 ℃ to 100 ℃ (T) ⁵ ) (t) of ⁵ ) From 1 hour to 24 hours) on the surface of the nanoparticle, which is functionalized with polyethylene glycol groups comprising ligands, or on the surface of the core-shell nanoparticle, which is functionalized with polyethylene glycol groups comprising ligandsThe alcohol group is functionalized. Optionally, a PEG-silane conjugate (PEG-silane ligand-free concentration between 10mM and 75 mM) (e.g., PEG-silane conjugate dissolved in a polar aprotic solvent such as DMSO or DMF) is then added to the resulting reaction mixture at room temperature, the reaction mixture comprising a nanoparticle surface functionalized with a polyethylene glycol group comprising a ligand or a core-shell nanoparticle surface functionalized with a polyethylene glycol group comprising a ligand, the resulting reaction mixture is maintained for a time (t) at ⁶ ) And temperature (T) ⁶ ) Lower (e.g. room temperature (T) ⁶ ) (t) of ⁶ ) From 0.5 minutes to 24 hours) (so that at least a portion of the PEG-silane conjugated molecule is adsorbed on at least a portion of the surface of the nanoparticle functionalized with polyethylene glycol groups comprising a ligand or on at least a portion of the surface of the core-shell nanoparticle functionalized with polyethylene glycol groups comprising a ligand), and for a time (t: (t) h) ⁷ ) And temperature (T) ⁷ ) Heating the resulting mixture (e.g. at 40 ℃ to 100 ℃ (T) ⁷ ) (t) of ⁷ ) From 1 hour to 24 hours) to form a nanoparticle surface functionalized with polyethylene glycol groups and ligand-containing polyethylene glycol groups or a core-shell nanoparticle surface functionalized with polyethylene glycol groups and ligand-containing polyethylene glycol groups.

In another example, at least a portion or all of the PEG-silane has a reactive group on a terminus of the PEG group opposite the terminus of the silane group conjugated to the PEG-silane conjugate (formed from the heterobifunctional PEG compound) and is functionalized with a polyethylene glycol group having a reactive group and optionally a polyethylene glycol group after formation of the nanoparticle surface or the surface of the core-shell nanoparticle functionalized with a polyethylene glycol group having a reactive group. Optionally, reacting a polyethylene glycol group with a second ligand functionalized with a second reactive group (which may be the same or different from the reactive group on the surface of the nanoparticle or the surface of the core-shell nanoparticle, the surface of the nanoparticle functionalized with a polyethylene glycol group and a polyethylene glycol group comprising a ligand, the surface of the core-shell nanoparticle functionalized with a polyethylene glycol group and a polyethylene glycol group comprising a ligand), the second ligand (which may be the same or different from the ligand on the surface of the nanoparticle or the surface of the core-shell nanoparticle, the surface of the nanoparticle functionalized with a polyethylene glycol group and a polyethylene glycol group comprising a ligand, the surface of the core-shell nanoparticle functionalized with a polyethylene glycol group and a polyethylene glycol group comprising a ligand), thereby forming a nanoparticle surface functionalized with polyethylene glycol groups functionalized with a second ligand and optionally polyethylene glycol groups, a core-shell nanoparticle surface functionalized with polyethylene glycol groups functionalized with a second ligand and polyethylene glycol groups and optionally polyethylene glycol groups.

In another example, at least a portion or all of the PEG-silane has a reactive group on a terminal end of the PEG group opposite to the terminal end of the silane group conjugated to the PEG-silane conjugate (formed from the heterobifunctional PEG compound) and, after formation of the nanoparticle surface or core-shell nanoparticle surface (the nanoparticle surface functionalized with an optional polyethylene glycol group having a reactive group and an optional polyethylene glycol group, the core-shell nanoparticle surface functionalized with a polyethylene glycol group having a reactive group and an optional polyethylene glycol group), is functionalized with a second reactive group (which may be the same or different from the reactive group of the nanoparticle surface or core-shell nanoparticle surface, the nanoparticle surface functionalized with a polyethylene glycol group and a polyethylene glycol group comprising a ligand), the surface of the core-shell nanoparticle functionalized with a polyethylene glycol group and a polyethylene glycol group comprising a ligand) reacting a second ligand (which may be the same or different from the ligand on the surface of the nanoparticle or the surface of the core-shell nanoparticle functionalized with a polyethylene glycol group and a polyethylene glycol group comprising a ligand), the surface of the nanoparticle being functionalized with a polyethylene glycol group and a polyethylene glycol group comprising a ligand), thereby forming a surface of the nanoparticle or a surface of the core-shell nanoparticle surface functionalized with a polyethylene glycol group functionalized with a second ligand and optionally a polyethylene glycol group, the surface of the core-shell nanoparticle being functionalized with a polyethylene glycol group functionalized with a second ligand and a polyethylene glycol group, and optionally functionalized with polyethylene glycol groups, wherein at least a portion of the PEG groups of the PEG-silane have reactive groups on the ends thereof opposite the ends of the silane groups conjugated to the PEG-silane conjugate (formed from the heterobifunctional PEG compound) and following formation of the following nanoparticle surface or core-shell nanoparticle surface: a nanoparticle surface functionalized with a polyethylene glycol group having a reactive group, a core-shell nanoparticle surface functionalized with a polyethylene glycol group having a reactive group, a nanoparticle surface functionalized with a polyethylene glycol group having a reactive group and a polyethylene glycol group containing a ligand, or a core-shell nanoparticle surface functionalized with a polyethylene glycol group having a reactive group and a polyethylene glycol group containing a ligand, the reactive group being reacted with a second ligand functionalized with a polyethylene glycol group and a polyethylene glycol group containing a ligand, the nanoparticle surface being functionalized with a polyethylene glycol group and a polyethylene glycol group containing a ligand, the second ligand being the same as or different from the ligand on the nanoparticle surface or the core-shell nanoparticle surface, the core-shell nanoparticle surface being functionalized with a polyethylene glycol group and a polyethylene glycol group containing a ligand, thereby forming a nanoparticle surface functionalized with polyethylene glycol groups and polyethylene glycol groups functionalized with a second ligand, a core-shell nanoparticle surface functionalized with polyethylene glycol groups and polyethylene glycol groups functionalized with a second ligand, a nanoparticle surface functionalized with polyethylene glycol groups comprising a ligand, or a core-shell nanoparticle surface functionalized with polyethylene glycol groups and polyethylene glycol groups comprising a ligand functionalized with a second ligand.

The nanoparticles may have a narrow size distribution. In various examples, the size distribution of the nanoparticles (before or after pegylation) is +/-5, 10, 15, or 20% of the average particle size (e.g., longest dimension) without including extraneous materials such as unreacted reagents, dust particles/aggregates. Particle size may be determined by methods known in the art. For example, particle size is determined by TEM, GPS or DLS. DLS contains systematic biases, so the DLS size distribution may not correlate with the size distribution measured by TEM or GPS.

The nanoparticles may comprise one or more groups derived from dye molecules other than functional groups (e.g., 1 to 7 dyes per nanoparticle). For example, a dye molecule or derivative of a dye molecule described herein is covalently bound to the network system of nanoparticles (e.g., via a linker moiety, which may be part of a dye precursor). The resulting covalently bound dye groups are derived from the original dye molecules. Illustrative, non-limiting examples of groups derived from dye molecules are described herein. In one example, the dye is incorporated into the silica or aluminosilicate network system using a dye precursor comprising a mixture with a sol-gel silica precursor (e.g., -Si (OR)) ₃ Group wherein R is alkyl) conjugated dye.

In one aspect, the present disclosure provides a composition comprising a nanoparticle of the present disclosure. The composition may comprise one or more types (e.g., having different average sizes and/or one or more different compositional characteristics). The compositions can include functionalized nanoparticles having one to five (e.g., 1, 2, 3, 4, or 5) types of different functional ligands located on (e.g., covalently bound to) the surface of the NP. (e.g., a pentafunctional nanoparticle (e.g., C' site) integrates multiple properties into a single NP, namely fluorescence detection, specific cell targeting, radioisotope chelation/labeling, ratiometric pH sensing, and drug delivery, while the overall NP size remains below 7 nm).

For example, the composition comprises a plurality of core and/or core-shell nanoparticles (e.g., silica core nanoparticles, silica core-shell nanoparticles, aluminosilicate core-shell nanoparticles). Any of the nanoparticles can be surface functionalized with one or more types of polyethylene glycol groups (e.g., polyethylene glycol groups, functionalized (e.g., functionalized with one or more ligands and/or reactive groups) polyethylene glycol groups, and combinations thereof). Any of the nanoparticles may have a dye or combination of dyes (e.g., NIR dyes) encapsulated therein. The dye molecule is covalently bound to the nanoparticle. Nanoparticles can be made by the methods described in this disclosure.

The nanoparticles in the composition can have a variety of sizes. The nanoparticles may have a core size of 2 to 15nm, including all values of 0.1nm and ranges therebetween. In various examples, the core of the nanoparticle has a size of 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, or 15 nm. In various examples, at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% of the nanoparticles (e.g., core and/or core-shell nanoparticles) have a size (e.g., longest dimension) of 2 to 15 nm. The nanoparticles may be ultra-small nanoparticles. In various examples, the ultra-small nanoparticles (e.g., ultra-small core and/or core-shell nanoparticles) have a size of 10nm or less (e.g., 2-8nm or 2-7 nm). In various examples, at least 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% of the ultra-small nanoparticles (e.g., ultra-small core and/or core-shell nanoparticles) have a size (e.g., longest dimension) of 10nm or less (e.g., 2-8nm or 2-7 nm). For exemplary size distributions, the composition may be subjected to no particle size differentiation (particle size selection/removal) process (e.g., filtration, dialysis, chromatography (e.g., GPC), centrifugation, etc.). For example, the nanoparticles described in the present disclosure are the only nanoparticles in the composition.

The composition may comprise other components. For example, the composition can further comprise a buffer suitable for administration to an individual (e.g., a mammal, such as a human). The buffer may be a pharmaceutically acceptable carrier.

The composition can have nanoparticles, particles (e.g., 2-15nm), dust particles/aggregates (>20nm), unreacted reagents (<2nm) at the time of synthesis and prior to any post-synthesis processing/handling.

In one aspect, the present disclosure provides uses of the nanoparticles and compositions described in the present disclosure. For example, the nanoparticles or compositions comprising the nanoparticles are used in therapeutic (e.g., delivery) and/or diagnostic (e.g., imaging) methods.

The ligand (functional group) carried by the nanoparticle may include a diagnostic agent and/or a therapeutic agent (e.g., a drug). Examples of therapeutic agents include, but are not limited to, chemotherapeutic agents, antibiotics, antifungal agents, antiparasitic agents, antiviral agents, and combinations thereof. Affinity ligands may also be conjugated to the nanoparticles to allow targeted delivery of the nanoparticles. For example, the nanoparticles may be conjugated to ligands that are capable of binding cellular components associated with a particular cell type (e.g., components on the cell membrane or in the intracellular compartment). The targeted molecule may be a tumor marker or a molecule in the signaling pathway. The ligand may have specific binding affinity for certain cell types (e.g., tumor cells). In certain examples, the ligand can be used to direct the nanoparticle to a specific region, such as the liver, spleen, brain, and the like. Imaging can be used to determine the location of the nanoparticles in the individual.

The nanoparticles or compositions comprising the nanoparticles may be administered to an individual, for example, in a pharmaceutically acceptable carrier that facilitates transport of the nanoparticles from one organ or portion of the body to another organ or portion of the body. Examples of individuals include animals, such as humans and non-human animals. Examples of individuals also include mammals.

The pharmaceutically acceptable carrier is typically water soluble. Some examples of materials that can be used in a pharmaceutically acceptable carrier include sugars such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; tragacanth powder; malt; gelatin; talc powder; excipients, such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; ringer's solution; ethanol; a phosphate buffer solution; and other non-toxic compatible materials used in pharmaceutical formulations. (see REMINGTON' S PHARM. SCI., 15 th edition (Mack Publ. Co., Easton (1975)) for example, other non-toxic suitable carriers or excipients may include buffers such as acetate, Tris, phosphate, citrate, and other organic acids, antioxidants including ascorbic acid and methionine, preservatives such as octadecyl dimethyl benzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol, or benzyl alcohol, alkyl parabens such as methyl or propyl parabens, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins, chelating agents such as EDTA, tonicity agents (toniifiers), such as trehalose and sodium chloride; sugars, such as sucrose, mannitol, trehalose, or sorbitol; surfactants, for example, polysorbates; salt-forming counterions, such as sodium; and/or a non-ionic surfactant, such as tween or polyethylene glycol (PEG). The pharmaceutical composition may comprise other therapeutic agents.

Compositions comprising the nanoparticles described herein can be administered to an individual by any suitable route, alone or in combination with other agents. Administration may be accomplished by any means, such as by parenteral, mucosal, pulmonary, topical, catheter-based, or oral delivery. Parenteral delivery may include, for example, subcutaneous, intravenous, intramuscular, intraarterial, and injection into organ tissue. Mucosal delivery may include, for example, intranasal delivery. Pulmonary delivery may include inhalation of the agent. Catheter-based delivery may include delivery through an iontophoresis-based catheter. Oral delivery may include the delivery of enteric coated pellets, or the administration of oral liquids. Transdermal delivery may include delivery through the use of a skin patch.

Following administration of a composition comprising nanoparticles described herein, one or more imaging techniques can be used to monitor the pathway, location, and clearance of the NPs. Examples of suitable imaging techniques include Artemis fluorescence camera systems.

The present disclosure provides a method of imaging a biological material (e.g., a cell, an extracellular component, or a tissue) comprising contacting the biological material with a nanoparticle comprising one or more dyes or a composition comprising the nanoparticle; directing exciting electromagnetic (e/m) radiation (e.g., light) onto the tissue or cell, thereby exciting the dye molecules; detecting e/m radiation emitted by the excited dye molecules; and capturing and processing the detected e/m radiation to provide one or more images of the biological material. One or more of these steps may be performed in vitro or in vivo. For example, the cell or tissue may be present in an individual or may be present in culture. Exposure of the cells or tissues to e/m radiation can be performed in vitro (e.g., under culture conditions) or in vivo. To direct e/m radiation to cells, extracellular material, tissue, organs, etc. within the individual or to any part of the individual's body that is not readily accessible, fiber optic instruments may be used.

For example, a method for imaging a region within an individual includes: (a) administering to the individual a nanoparticle or composition of the present disclosure comprising one or more dye molecules; (b) directing excitation light into the subject, thereby exciting at least one of the one or more dye molecules; (c) detecting the excited light, the detected light being emitted by the dye molecules in the individual as a result of excitation by the excitation light; and (d) processing signals corresponding to the detected light to provide one or more images (e.g., a real-time video stream) of a region within the object.

Since fluorescent particles are brighter than free dye, fluorescent particles can be used for tissue imaging as well as imaging metastases. Additionally or alternatively, the radioisotope may be further attached to ligand groups (e.g., tyrosine residues or chelating agents) of ligand functionalized particles or to a silica matrix of pegylated particles without specific ligand functionalization for light-induced electron transfer imaging. If a radioisotope is selected for treatment, e.g. ²²⁵ Ac or ¹⁷⁷ Lu, which in turn will result in the particles having additional radiotherapeutic properties.

For example, a drug-linker conjugate in which the linker group can be specifically cleaved by an enzyme or acidic conditions in a tumor to release the drug can be covalently attached to a functional ligand on the particle for drug delivery. For example, the drug-linker-thiol conjugate can be attached to the maleimido-PEG-particle by a thiol-maleimido conjugation reaction after the synthesis of the maleimido-PEG-particle. In addition, both drug-linker conjugates and cancer targeting peptides can be attached to the surface of the particles to deliver the drug specifically to the tumor.

The steps of the methods described in the various embodiments and examples disclosed herein are sufficient to practice the methods and produce the compositions described in the present disclosure. Thus, in one embodiment, the method consists essentially of a combination of the steps of the methods disclosed herein. In another embodiment, the method consists of this step.

In the following statements, various embodiments of the methods and compositions of the present disclosure, as well as methods of using the compositions, are described:

statement 1. a method of forming a functionalized nanoparticle of the present disclosure, the method comprising:

contacting a nanoparticle, e.g., a silica nanoparticle or an aluminosilicate particle, (e.g., having a size (e.g., longest dimension) of 2 to 15nm (e.g., 10nm or less, such as 2 to 10nm or 2 to 9.99nm)) with one or more functionalized precursors, the nanoparticle comprising a plurality of polyethylene glycol (PEG) groups covalently bound to a surface of the nanoparticle, the one or more functionalized precursors comprising at least one reactive group, wherein a functionalized nanoparticle is formed, the functionalized nanoparticle comprising at least one reactive group covalently bound to a surface of the functionalized nanoparticle.

Statement 2. the method of statement 1, wherein the contacting is carried out in an aqueous medium (e.g., water).

Statement 3. the method of statement 1 or 2, wherein if the nanoparticle is contacted with two or more functionalized precursors (e.g., 2, 3, 4, or 5 functionalized precursors, each having at least one reactive group, wherein at least two or all of the reactive groups of each individual functionalized precursor are structurally different from the reactive groups of the other functionalized precursors), the contacting is performed in a single reaction mixture.

Statement 4. the method of any of the preceding statements, wherein, if the nanoparticle is contacted with two or more functionalized precursors (e.g., 2, 3, 4, or 5 functionalized precursors, each having at least one reactive group, wherein at least two or all of the reactive groups of each individual functionalized precursor are structurally different from the reactive groups of the other functionalized precursors), the contacting is performed in at least two different reaction mixtures.

Statement 5. the method of any of the preceding statements, further comprising: contacting the functionalized nanoparticles with at least one further functionalized precursor comprising at least one, at least two reactive groups, all reactive groups of each individual functionalized precursor being structurally different from the reactive groups of the other functionalized precursor groups, wherein the functional group of the at least one additional functionalized precursor is structurally different from the reactive group of the functionalized precursor, and where the functionalized nanoparticles are contacted with two or more additional functionalizing precursors, each individual functionalizing precursor reactive group is structurally different from the reactive groups of the other functionalizing precursors, and forming a functionalized nanoparticle comprising two or more reactive groups covalently bound to the surface of the functionalized nanoparticle.

Statement 6. the method of any of the preceding statements, further comprising contacting the functionalized nanoparticles with one or more functional group precursors comprising one or more functional groups, wherein functionalized nanoparticles comprising the functional groups are formed and each functional group is covalently bound to the surface of the functionalized nanoparticles.

Statement 7. the method of statement 6, wherein the contacting of each functional group precursor is performed in a single reaction mixture.

Statement 8. the method of statement 6, wherein for each contacting of a separate functional group precursor, the contacting is performed in a separate reaction mixture.

Statement 9. the method according to any of the preceding statements, wherein the nanoparticle surface functionalized with polyethylene glycol (PEG) is contacted with a first functionalized precursor comprising at least one first reactive group, wherein a first functionalized nanoparticle comprising the at least one reactive group covalently bound to the surface of the functionalized nanoparticle is formed.

Statement 10. the method of statement 9, further comprising contacting the functionalized nanoparticle with a second functionalizing precursor comprising at least one second reactive group, wherein the first reactive group and the second reactive group are structurally different, wherein a second functionalized nanoparticle is formed comprising the at least one first reactive group and the at least one second reactive group, each of which is respectively covalently bound to the surface of the functionalized nanoparticle.

Statement 11. the method of statement 10, further comprising contacting the second functionalized nanoparticle with a third functionalized precursor comprising at least one third reactive group, wherein the first, second, and third reactive groups are structurally different from one another, wherein a third functionalized nanoparticle is formed comprising the at least one first, second, and third reactive groups, each of which is covalently bound to the surface of the functionalized nanoparticle, respectively.

Statement 12. the method of statement 11, further comprising contacting the third functionalized nanoparticle with a third functionalized precursor comprising at least one third reactive group, wherein the first reactive group, the second reactive group, and the third reactive group are structurally different from one another, wherein a third functionalized nanoparticle is formed comprising the at least one first reactive group, the at least one second reactive group, and at least one third reactive group, each of which is covalently bound to the surface of the functionalized nanoparticle, respectively.

Statement 13. the method of statement 12, further comprising contacting the third functionalized nanoparticle with a fourth functionalized precursor comprising at least one fourth reactive group, wherein the first reactive group, the second reactive group, the third reactive group, and the fourth reactive group are structurally different from one another, wherein a fourth functionalized nanoparticle is formed comprising the at least one first reactive group, the at least one second reactive group, and the at least one third reactive group, and the at least one fourth reactive group, each of which is covalently bound to a surface of the functionalized nanoparticle, respectively.

Statement 14. the method of statement 13, further comprising contacting the fourth functionalized nanoparticle with a fifth functionalized precursor comprising at least one fifth reactive group, wherein the first reactive group, the second reactive group, the third reactive group, the fourth reactive group, and the fifth reactive group are structurally different from one another, wherein a fifth functionalized nanoparticle is formed comprising the at least one first reactive group, the at least one second reactive group, the at least one third reactive group, the at least one fourth reactive group, and the at least one fifth reactive group, each of which is covalently bound to a surface of the functionalized nanoparticle, respectively.

Statement 15. the method of any of statements 9-14, further comprising contacting the functionalized nanoparticles with one or more functional group precursors (e.g., a first functional group precursor, a second functional group precursor, a third functional group precursor, a fourth functional group precursor, a fifth functional group precursor, or a combination thereof) comprising one or more functional groups, wherein functionalized nanoparticles comprising the functional groups are formed and each functional group is covalently bound to the surface of the functionalized nanoparticles.

Statement 16. the method of statement 15, wherein the contacting of each functional group precursor is performed in a single reaction mixture.

Statement 17. the method of statement 15, wherein the contacting is performed in a separate reaction mixture for each contacting of a single functional group precursor.

Statement 18. the method of any of the preceding statements, wherein the functionalized precursor comprises a reactive group selected from: amine groups, thiol groups, carboxylic acid groups, carboxylate groups, ester groups (e.g., activated ester groups), maleimide groups, allyl groups, terminal alkyne groups, azide groups, thiocyanate groups, and combinations thereof.

Statement 19. the method of any of the preceding statements, wherein the functionalized group precursor comprises one or more silane groups and one or more reactive groups.

Statement 20. the method of any of the preceding statements, wherein the silane groups are selected from the following:

and combinations thereof, wherein R is independently at each occurrence alkyl (e.g., C) ₁ 、C ₂ 、C ₃ Or C ₄ Alkyl) andand R is independently at each occurrence H or alkyl (e.g., C) ₁ 、C ₂ 、C ₃ Or C ₄ Alkyl groups).

The method of any of the preceding statements, wherein one or more of the functionalized precursors has the structure:

wherein X is a reactive group described herein (e.g., an amine group, a thiol group, a carboxylic acid group, a carboxylate group, an ester group (e.g., an activated ester group), a maleimide group, an allyl group, a terminal alkynyl group, an azido group, or a thiocyanate group), n is, e.g., 1, 2, 3, 4, 5, 6, 7, or 8, and R is independently at each occurrence an alkyl group (e.g., C ₁ 、C ₂ 、C ₃ Or C ₄ Alkyl).

Statement 22. the method of any of statements 6-8 or 15-20, wherein at least one functional group or all of the functional groups are covalently bound to the surface of the nanoparticle through a linking group.

Statement 23. the method of statement 22, wherein in each individual case, the linking group is selected from alkyl (e.g., C) ₁ 、C ₂ 、C ₃ Or C ₄ Alkyl groups).

Statement 24. the method of any of statements 6-8 or 15-20, wherein the functional group is selected from the group consisting of the functional groups described herein and combinations thereof.

Statement 25. a composition comprising a plurality of nanoparticles described herein (e.g., a plurality of nanoparticles, each nanoparticle comprising a plurality of polyethylene glycol (PEG) groups covalently bound to a surface of the nanoparticle and at least one functional group (e.g., 1, 2, 3, 4, or 5 different types of functional groups) covalently bound to a surface of the functionalized nanoparticle), wherein at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%, or 100%) of the nanoparticles have a size described herein (e.g., a size of 2 to 15nm (e.g., 10nm or less, e.g., 2-8 or 2-7nm), or 2 to 10nm or 2 to 9.99nm)), and the composition has not been subjected to any size differentiation process.

Statement 26. the composition of statement 25, wherein the nanoparticle is a core nanoparticle (e.g., having a size (e.g., longest dimension) of 2 to 15nm (e.g., 10nm or less, such as 2 to 10nm or 2 to 9.99nm)), a core-shell nanoparticle (e.g., having a size (e.g., longest dimension) of 2 to 15nm (e.g., 10nm or less, such as 2 to 10nm or 2 to 9.99nm)), or a combination thereof.

Statement 27. the composition of statement 26, wherein the core nanoparticle is an aluminosilicate core nanoparticle or a silica core nanoparticle.

Statement 28. the composition of statement 26 or 27, wherein the core is a silica core, or the core and the shell of the core-shell nanoparticle are silica shells.

Statement 29. the composition of any of statements 26-28, wherein the core of the core-shell nanoparticles is an aluminosilicate core and the shell of the core-shell nanoparticles is a silica shell.

Statement 30. the composition of any of statements 25-29, wherein the functional group is selected from the group consisting of the functional groups described herein and combinations thereof.

Statement 31. the method of any one of statements 25-30, wherein at least one or all of the functional groups are covalently bound to the surface of the nanoparticle through a linking group.

Statement 32. the method of statement 31, wherein in each individual case, the linking group is selected from alkyl (e.g., C) ₁ To C ₄ Alkyl groups).

Statement 33. the composition of any of statements 25-32, wherein 1 to 100 (e.g., 20 to 100, 25 to 100, 30 to 100, 35 to 100, 40 to 100, or 50 to 100) functional groups (e.g., an average of 1 to 100 functional groups) are covalently bound to the surface of each of the nanoparticles.

Statement 34. the composition of any of statements 25-33, wherein at least a portion or all of the polyethylene groups comprise one or more functional groups.

Statement 35. the method of statement 34, wherein the functional group is selected from the group consisting of the functional groups described herein and combinations thereof.

Statement 36. the composition of any one of statements 25-35, wherein the nanoparticle further comprises one or more dye molecules described herein, or a combination thereof, encapsulated therein.

Statement 37. the composition of statement 36, wherein the number of dye molecules per core is from 1 to 7.

Statement 38. the composition of any of statements 25-37, wherein the composition is stable (e.g., no aggregation and/or decomposition (e.g., loss of functional groups) is observed by Gel Permeation Chromatography (GPC) or a combination of Fluorescence Correlation Spectroscopy (FCS) and GPC) (e.g., stable for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, or 24 months).

Statement 39. a diagnostic method (e.g., imaging method) disclosed herein using a composition according to any of statements 25-38 (e.g., a method for imaging a region within an individual disclosed herein, the method comprises the following steps: administering to the individual a composition according to any of statements 25-38, wherein the nanoparticle comprises one or more dye molecules and/or one or more dye groups; an excitation electromagnetic radiation is directed into the object, thereby exciting at least one of the one or more dye molecules and/or one or more dye groups; the excited electromagnetic radiation is detected and, the detected electromagnetic radiation is emitted by the dye molecules in the individual as a result of excitation by the exciting electromagnetic radiation; and processing signals corresponding to the detected electromagnetic radiation to provide one or more images of a region within the object.

Statement 40. a method of treatment disclosed herein (e.g., a method of delivering a drug to an individual) using a composition according to any of statements 25-38, the composition comprising one or more nanoparticles having a functional group comprising a drug or a functional group derived from a drug (e.g., a method for delivering a drug to an individual disclosed herein) comprising administering to the individual a composition according to any of statements 25-38, the composition comprising one or more nanoparticles having a functional group comprising a drug or a functional group derived from a drug, wherein the drug is released within the individual.

Statement 41. the method of treatment according to statement 40, further comprising imaging a region within the individual as disclosed herein (e.g., the method according to statement 39).

The following examples are provided to illustrate the present disclosure. They are not intended to be limiting in any way.

Example 1

This example provides examples of the methods and compositions described in this disclosure and uses thereof.

In the fields of nanobiotechnology and nanomedicine, multifunctional Nanoparticles (NPs) that combine different functional components into a single NP platform are of great interest. In this example, we describe a general surface modification method to modularly and orthogonally functionalize Cornell prime dots (C' dots), i.e., ultra-small pegylated fluorescent core-shell silica nanoparticles under 10nm, with up to four different types of functional ligands on the NP surface. It is capable of synthesizing five-functional C' spots that integrate multiple properties (i.e., fluorescence detection, specific cell targeting, radioisotope chelation/labeling, ratiometric pH sensing, and drug delivery) into a single NP while the overall NP size is still below 7 nm. This is achieved by exploiting the fact that the PEG layer at the C' site can be penetrated by small molecules. Amine and/or thiol functionalized silane molecules can be inserted between PEG chains and on the silica surface at the C' point, and additional functional ligands can then be attached to the amine and/or thiol functionalized silane molecules. This method of modifying the PEGylation back surface (PPSMI) by insertion requires only a few additional steps between PEGylation and purification at the C' point in a one-pot water-based synthesis, while not reducing the production of high quality NPs. The resulting C' dots with additional functionality show physicochemical properties such as their size and PEG density close to the clinically transformed C-dots, opening the door for diversification of their clinical applications. We also demonstrated that the modification of the C' point synthesis allows for a large number of targeting peptides per particle, and that a simple and versatile spectroscopic method quantitatively assesses the specific number of different surface ligands by deconvoluting the absorption spectra into individual components. The experience gained from this study of synthetic pegylation and post-pegylation surface modification methods can be transferred to the development of other pegylation NP platforms for biomedical applications and clinical transformations.

In this example, we describe a modular and orthogonal approach to modifying the pegylation back surface (PPSMI) by insertion, which approach is able to achieve this goal (fig. 1). It consists of the following: in the synthesis step performed between NP pegylation and purification, silanes with orthogonal functional groups (e.g., amines and thiols) were covalently inserted between the PEG chains and on the silica surface at the C' point (fig. 1). We demonstrate that in this way the limitations of existing functionalization strategies, including particle aggregation observed when introducing these groups during pegylation, can be overcome. The PPSMI process retains the one-pot nature of C' site synthesis in aqueous media. Furthermore, we will show that these modular and orthogonal surface modification reactions have only a minor impact on the overall physicochemical properties of the particles (including size and PEG density), thereby maximizing the chances of their clinical transformation. By using this method, various multifunctional C' points can be generated (table 1). In particular, pentafunctional C' dot particles were synthesized with a total of four functional ligands attached to the NP surface, allowing simultaneous fluorescent tracking, tumor targeting, ratiometric pH sensing, radioisotope chelation, and disease treatment (fig. 1). This surface modification method utilizes the following facts: the PEG layer of a well-pegylated NP is still penetrable by other molecules. To properly illustrate the chemical complexity of such particles, and to distinguish different multifunctional NP chemistries from each other, we developed a naming system that contains information on: (i) NP platforms, e.g. C' dots compared to mesoporous C dots (mC dots), (ii) encapsulated fluorescent dyes, (iii) specific surface functionalities and their connectivity to the particles, (iv) specific attachment chemistries, (v) specific PEG chain lengths, etc. (e.g. fig. 7). We finally demonstrated a spectroscopic method that quantitatively assessed the specific number of different ligands introduced to the particle surface by deconvoluting the absorption spectrum into individual components. We hope that the experience gained in this study will also open the way for the development of other pegylated NP platforms in biomedical applications.

Results and discussion. C, (RGDyC) -PEG-Cy5.5-C' point synthesis and purification. The previously reported synthesis of the C 'site is a versatile method that enables the functionalization of the C' site with different types of dyes and cancer targeting peptides (fig. 8). For example, to generate the NIR dye Cy5.5 and target alpha _v β ₃ The C' site of the cyclic (arginine-glycine-aspartic acid-D-tyrosine-cysteine) peptide (C (rgdyc)) functionalization of integrins, which is referred to as the C (rgdyc) -PEG-cy5.5-C site, Tetramethylorthosilicate (TMOS) and cy5.5 silane conjugate (cy5.5-silane) were first added to an aqueous ammonium hydroxide solution at pH of about 8 at room temperature. The hydrolyzed TMOS and cy5.5-silane molecules are condensed together to form ultra-small silica NPs, in which the cy5.5 dye is covalently encapsulated in a silica matrix. In the next step, silane-functionalized PEG (PEG-silane) and PEG-silane modified with c (rgdyc) peptide (c (rgdyc) -PEG-silane) are added together to the reaction mixture. After addition, c (rgdyc) -PEG-silane and PEG-silane rapidly adsorb on the NP surface through hydrogen bonding between the surface silanol groups and PEG, thereby terminating nanoparticle growth. After allowing the reaction mixture to stand at room temperature overnightThe reaction temperature was raised to 80 ℃ and held at this temperature overnight to enhance covalent attachment of PEG-silane and c (rggyc) -PEG-silane to the NP surface. The reaction mixture was then cooled to room temperature and finally purified using Gel Permeation Chromatography (GPC) at the C (rgdyc) -cy5.5-C point, followed by filtration through sterile filters for final use.

The GPC elution pattern of the (RGDyC) -PEG-Cy5.5-C' spot showed three peaks before purification (FIG. 2A and FIG. 8A). The major peak at about 9 minutes corresponds to the C (rgdyc) -PEG-cy5.5-C' point nanoparticle product, while the peaks at about 5 minutes and 16 minutes correspond to impurities from the PEG reagent and unreacted cy5.5-silane, respectively. The elution volume corresponding to the nanoparticle product was collected and run through the GPC column again to ensure the highest purity. After GPC purification, the elution profile of the final C point solution showed only a single peak around 9 minutes, indicating a GPC purity of about 100% (fig. 8B). Furthermore, the product peaks show very narrow and highly symmetric shapes with no detectable skew and can be fitted using a single component gaussian distribution, which indicates that the NP product has a very narrow particle size distribution.

The purified C (RGDyC) -PEG-Cy5.5-C' spots were then characterized by absorbance and Fluorescence Correlation Spectroscopy (FCS). The absorption spectrum of the C (RGDyC) -PEG-Cy5.5-C ' point shows a peak at about 675nm corresponding to Cy5.5 dye and a small peak at about 275nm (FIG. 8D), whereas the PEGylated Cy5.5-C ' point without C (RGDyC) functionalization (PEG-Cy5.5-C ' point) does not. Thus, the presence of this peak is characteristic of the C (rgdyc) peptide, indicating that C (rgdyc) was successfully attached to the C' dot surface. The FCS curve at the C (rgdyc) -PEG-cy5.5-C' point was well fitted using a monomodal FCS correlation function, confirming that the NP product has a very narrow particle size distribution (fig. 8C). By FCS fitting, the hydrated particle size and particle concentration can be determined. The amount of the cy5.5 dye and C (rgdyc) ligand per C-point can be estimated by dividing the concentration of cy5.5 and C (rgdyc), respectively, obtained from the absorbance measurements by the particle concentration obtained from FCS. The results show that the average diameter of the C (RGDyC) -PEG-Cy5.5-C' site is about 6.4nm, and there are about 1.6 Cy5.5 dyes and 24C (RGDyC) peptides per NP (Table 1). Finally, the brightness of the particles can also be estimated by combining FCS and absorbance measurements, which indicates that the C (rgdyc) -PEG-cy 5.5-C' point in aqueous solution is about three times brighter than the free cy5.5 dye under the excitation conditions set by the FCS. The final C (RGDyC) -PEG-Cy5.5-C' point product was stored in Deionized (DI) water at 4 ℃ for further use.

The c (rgdyc) surface ligand density was increased by adjusting the pegylation conditions. In the previously reported synthetic method of clinical transformation of C' spots, in the pegylation step, C (rgdyc) cancer targeting peptides were introduced by condensing both C (rgdyc) -PEG-silane and PEG-silane onto the NP surface. Considering that c (rgdyc) -PEG-silane is much more expensive than PEG-silane, c (rgdyc) -PEG-silane is always added to the reaction mixture before PEG-silane is added, enabling reaction with the original NP surface to achieve the desired conversion efficiency. Although this approach can be used to generate C 'spots with up to about 20-25C (rgdyc) ligands per NP, we observed that further boosting the concentration of C (rgdyc) -PEG-silane did not generate narrowly distributed C' spots. Instead, the synthesis mixture became cloudy immediately after the addition of these high concentrations of c (rgdyc) -PEG-silane, indicating aggregation of the NP. In fact, the GPC elution profile of the reaction product showed a broad peak between 5-11 minutes due to NP aggregation (fig. 2B). Even after an infinite number of cycles of GPC purification, the GPC elution profile of the NP product is still broad, indicating a synthesis failure.

We found that the amount of c (RGDyC) -PEG-silane acceptable in the synthesis was comparable to ammonium hydroxide [ NH3OH ] without causing NP aggregation]Is highly sensitive. If [ NH ] ₃ OH]From the standard 2mM up to 6mM, the c (RGDyC) -PEG-silane concentration can be tripled while maintaining good NP product quality. In [ NH ] ₃ OH]The GPC elution profile at C' point, 6mM and synthesized at three times the C (rgdyc) -PEG-silane concentration (fig. 2C) shows similar characteristics as under conventional conditions (fig. 2A). The results indicate a high quality NP product that enables separation of C' points from other impurities by using GPC as previously described. After purification, a narrow distribution of C (RGDyC) -PEG-Cy5.5-C' spots was successfully generated, with an estimated 58 ligands per NP (C: (RGDyC))Fig. 2D to F). As expected, the absorption at 275nm in the UV-vis spectrum was enhanced compared to the C (RGDyC) -PEG-Cy5.5-C' point with lower ligand numbers (FIG. 2F). According to FCS characterization, these particles also have slightly increased hydrated particle size compared to particles with lower ligand numbers (i.e. 24c (rgdyc)'s), i.e. 6.8nm compared to 6.4nm (fig. 2E and table 1), which is likely due to the increased c (rgdyc) number per particle (fig. 2D), rather than to the larger silica core.

For successful c (RGDyC) surface functionalization, p [ NH3OH]May be due to the rapid adsorption of c (rgdyc) peptides to bare silica surfaces and resulting changes in electrostatic interactions between NPs. For the C' spot synthesis, it has been demonstrated that both PEG and PEG-peptide conjugates are rapidly adsorbed onto the bare silica NP surface, possibly through hydrogen bonding, during the pegylation step. This rapid PEG adsorption was used in the synthesis of the C' site to effectively terminate NP growth and increase the conversion efficiency of the C (rgdyc) -PEG-silane additive. However, since each c (rgdyc) peptide contains several amine groups that are positively charged at reaction pH, i.e., they are weak bases, adsorption of the c (rgdyc) peptide can effectively screen out the net negative charge of bare silica NPs, resulting in loss of electrostatic stability of the NPs, thereby leading to NP aggregation. In contrast, when [ NH ] ₃ OH]With a slight increase, the average net positive charge of the c (rgdyc) peptide is reduced, allowing the ultra-small silica NPs to adsorb more c (rgdyc) -PEG-silane during pegylation, and at the same time not lose NP stability. While more systematic studies are needed to confirm the hypothesis for this mechanism, the ability to increase the surface ligand density of cancer targeting peptides more than two-fold may further enhance the tumor targeting efficiency of the C' site.

Secondary functional groups are introduced to the surface of the C' dots during pegylation by co-condensing the ligand-PEG-silane conjugates. The reaction conditions developed for functionalizing C 'dot surfaces with up to 58C (rgdyc) ligands were also used to introduce various types of other functional ligands to the C' dot surfaces. This is achieved by replacing c (rgdyc) -PEG-silane with a combination of different ligand-PEG-silane and/or ligand-silane conjugates in the pegylation step. However, the quality of the synthesized multifunctional C' site, and therefore the success rate of using this method, is largely dependent on the characteristics of the functional ligand.

For example, the combination of C (rgdyc) -PEG-silane, maleimide (mal) functionalized PEG-silane (mal-PEG-silane) and PEG-silane were added in this order to the reaction mixture based on the C' point of Cy5 in the pegylation step. In addition to the C (rgdyc) peptide, the resulting C (rgdyc) -mal-PEG-Cy5-C 'site also contains a maleimide group on the surface of the pegylated NP, thereby enabling the C' site to be further reacted with, for example, a thiol-functionalized drug-linker conjugate for theranostic applications (fig. 1). Although the GPC elution profile of the C (rgdyc) -mal-PEG-Cy 5-C' spot at the time of synthesis (i.e., prior to GPC purification) showed a slightly skewed NP peak (fig. 9A), a symmetric NP peak was obtained after four cycles of GPC purification along with satisfactory FCS characterization results (fig. 9B and C), indicating that the quality of the NP synthesis product was acceptable. The absorption spectra of the purified C (rgdyc) -mal-PEG-Cy 5-C' spots obtained from different mal-PEG-silane concentrations showed not only a strong peak at about 275nm due to the C (rgdyc) ligand, but also an increased absorption below 250nm compared to the control NP, which we attributed to the presence of maleimide functional groups (fig. 9D). According to FCS, the hydrated particle size of the C (RGDyC) -mal-PEG-Cy 5-C' spot was about 6.8nm (Table 1).

When other types of ligands are used, the synthesis of multifunctional C' sites may not be equally successful. For example, the maleimide group is substituted with a Dibenzocyclooctyl (DBCO) group that can react with an azide group, allowing the C' site to be modified with other ligands by click chemistry. Although monodisperse DBCO-C (rgdyc) -PEG-C 'dots (fig. 10A to D, table 1) can be produced using this method according to GPC, UV-vis and FCS characterization, no reactivity of DBCO was observed when attempting to conjugate DBCO-C (rgdyc) -PEG-C' dots with azide functionalities. This indicates that the accessibility of the DBCO ligand on the surface of the C' spot is low. This, in turn, may be attributed to the increased hydrophobicity of the DBCO ligand compared to the maleimide group. The poor water solubility of DBCO ligands may cause them to remain associated with the C' point silica surface even after NP pegylation, limiting their accessibility and reactivity.

In our original experiment, the C (RGDyC) -PEG-Cy5-C site was labeled with the tyrosine residue of the C (RGDyC) peptide ¹²⁴ I, so that Positron Emission Tomography (PET) imaging can be used. To use other radioactive isotopes such as zirconium (b), (c), (d) and (d) b) ⁸⁹ Zr) or lutetium ¹⁷⁷ Lu) to mark the C 'spot, it is highly desirable to functionalize the C' spot with a specific chelator ligand. For example, Desferrioxamine (DFO) is directed to ⁸⁹ One of the most effective chelating agents for Zr radiolabelling. To functionalize the C' site with DFO, isothiocyanate (NCS) functionalized DFO (NCS-DFO) was first conjugated with an amino-silane to produce DFO-silane, which was then added to the reaction mixture during pegylation along with C (rgdyc) -PEG-silane and PEG-silane. Although narrow distribution of DFO-C (rgdyc) -PEG-C 'sites was generated by this method (fig. 10E to H and table 1), these NPs showed unfavorable results in biological experiments, which we temporarily attributed to the heterogeneous distribution of DFO groups in different C' sites (data not shown).

The last example is the functionalization of the C (rgdyc) -PEG-C 'site with amine groups, so that the C' site can be modified with amine reactive functional ligands. When an aminosilane, such as aminopropyl-trimethoxy-silane (APTMS), is introduced during PEGylation along with c (RGDyC) -PEG-silane and PEG-silane, the resulting c (RGDyC) -NH ₂ The PEG-C' point shows a broadened particle peak in the GPC elution profile (fig. 3A). The peak remained skewed even after four cycles of GPC purification (fig. 3B). The skewed GPC elution profile indicates aggregation of the NP, most likely due to the attached amine groups that catalyze the formation of Si-O-Si bonds, resulting in loss of NP stability. Although FCS characterization showed that the average particle size remained around 6.8nm after GPC purification (fig. 3C and table 1), since a parent aggregation peak was still observed for the purified NPs, it was shown that this NP aggregation greatly affected the yield and quality of the final C' point product (fig. 3B). In addition, the C (RGDyC) -NH synthesized using this method compared to the standard C (RGDyC) -PEG-Cy 5-C' spot ₂ The absorbance at the-PEG-Cy 5-C' spot showed an additional peak at about 480nm (FIG. 3D). This absorbanceThis could be attributed to degradation products of the Cy5 dye, probably due to the interaction between the amine group and the Cy5 molecule near the silica surface. This degradation of Cy5 dye reduced the fluorescence intensity of the particles and was very detrimental for biomedical applications.

The second functional group is introduced to the surface of the C' dot by insertion modification of the PEGylated rear surface (PPSMI). The challenge of introducing a second functional group to the C' spot surface by co-condensing a different ligand-silane conjugate during pegylation stems from the large variation in affinity between the different functional groups and the bare silica NP surface. Many functional ligands of interest in biomedical applications carry a large number of amine and/or hydroxyl groups, which have a high affinity for silica. As mentioned in the previous section, the strong association between such ligands and silica may lead to their poor accessibility and maldistribution.

One possible solution to this challenge is to control the kinetics of the association between the ligand and the silica. Ideally, it is desirable to have a degree of affinity between the ligand and the bare silica to increase the reaction conversion. On the other hand, too high an affinity may result in hindering the accessibility of the ligands and an uneven distribution of the ligands. Relying on controlled association kinetics is not an optimal solution for a number of reasons. First, the association between the ligand and the silica is highly sensitive to the details of the ligand molecular structure. Optimizing the pegylation conditions individually for each different ligand would greatly reduce efficiency. Second, since bare silica NPs in water are electrostatically stable and their stability is highly sensitive to reaction conditions including ionic strength and pH, adjusting pegylation conditions may interfere with the stability of the NPs, leading to synthesis failure (see above).

To overcome these problems, we did not continue to functionalize the C' site in the pegylation step, instead we attempted a reaction that modified the pegylation back surface (PPSMI) by insertion. This PPSMI method utilizes a mature C' point pegylation protocol. Pegylation of the particles slows the association of other ligands with the surface of the silica NPs, allowing more time for these ligands to be uniformly dispersed in the reaction mixture prior to reaction with the silica NPs. Furthermore, well-defined pegylation confers unique steric stability to the C' site, which protects it from aggregation even under more extreme synthetic conditions. Thus, it provides a broader window for adjusting reaction parameters, thereby enabling efficient surface modification with other ligands. Most importantly, it has recently been reported that the PEG layer of PEGylated NPs may still be penetrated by biomolecules such as proteins, depending on several parameters, including surface PEG density and NP surface curvature. At the same time, even after pegylation, silanol groups always remain on the interface between the silica core and the PEG layer of the pegylated silica nanoparticles, potentially enabling further condensation with small silane molecules.

To do this, we first tried adding DFO-silane to the reaction mixture at the C (RGDyC) -PEG-Cy 5-C' site after the PEGylation step. Unfortunately, the final product did not show the expected increase in absorbance signal below 300nm, indicating a less efficient reaction with DFO-silane (fig. 11). This is due to several reasons. First, because the molar mass of the DFO-silane conjugate is even greater than the PEG-silane used in the C' point pegylation, it may not diffuse efficiently through the PEG layer and react with the underlying silica surface silanol groups. This indicates that the PEG layer of the pegylated C' dot is quite dense, consistent with our previous studies. Second, the self-condensation of the hydrolyzed DFO-silane competes with its attachment to the C' site. Since permeation of DFO-silane through the PEG layer is slow, self-condensation is the predominant reaction. Based on these preliminary results and conclusions, we chose an alternative method to functionalize the C (rgdyc) -PEG-Cy 5-C' spot with DFO, where the surface modification is divided into two steps (fig. 1). In the first step, an amino-silane is added to the reaction mixture. The amino-silane is a relatively small molecule and therefore is expected to diffuse more rapidly into the PEG layer and attach to the underlying silica surface. The resulting NH was compared to the corresponding C (RGDyC) -PEG-Cy 5-C' spot ₂ The (rgdyc) -PEG-Cy5-C spot did not show any substantial change in particle characteristics (table 1, compare fig. 3E-H with fig. 8E-H additionally). Example (b)E.g., C (RGDyC) -PEG-Cy 5-C' dots and NH ₂ The absorption spectra of the (C) (rgdyc) -PEG-Cy5-C 'spot almost completely overlapped, indicating that the attachment of the amino-silane to the C (rgdyc) -PEG-Cy 5-C' spot did not have any effect on the optical characteristics of these particles (fig. 3H).

Additional surface amine groups can then be used to introduce second functional ligands to the surface of the C' point silica. To verify this idea, after the addition of the aminosilane, DFO-NCS was added in a subsequent step to react with amine groups located under the PEG layer via an amine group-NCS reaction (fig. 1). The advantage of this method is that a relatively high concentration of DFO can now be applied to push the DFO-NCS molecules through the PEG layer, while excess DFO can be easily removed by centrifugation and GPC in the final purification step. The resulting GPC elution pattern at the DFO-C (RGDyC) -PEG-Cy 5-C' spot showed that the sample was relatively clean with only one major peak of NP product (FIG. 4A). After purification, a narrow distribution of C' spots with a diameter of 6.4nm was obtained (FIGS. 4B and C, Table 1). And NH ₂ The purified DFO-C (RGDyC) -PEG-Cy5-C 'spot had a unique absorbance profile between 200 and 300nm compared to the (RGDyC) -PEG-Cy 5-C' spot (FIG. 4D). This absorbance signal corresponds to the absorbance of the DFO molecule and increases with increasing concentration of DFO-NCS used in the reaction. The absorbance at the DFO-C (RGDyC) -PEG-Cy 5-C' point was deconvoluted to the independent contribution of each fraction by fitting the spectra using a set of absorbance standards (FIG. 12). For the C' spot synthesized using DFO-NCS at a concentration of about 0.2mM, the numbers of Cy5, C (RGDyC) and DFO molecules per particle were estimated to be about 1.6, 23, and 4, respectively (FIG. 12 and Table 1). These spectral results indicate that DFO had successfully attached to NH2-C (RGDyC) -PEG-Cy 5-C' spot and confirmed NH ₂ The accessibility of the amine group at the (RGDyC) -PEG-Cy 5-C' site enables further reactions. In addition to the attachment of DFO molecules, relative to the C (RGDyC) -PEG-Cy 5-C' site or NH ₂ The C (rgdyc) -PEG-Cy5-C ' spot, no substantial change was observed in further GPC and FCS based C ' spot characterization (table 1), indicating that this modification after pegylation at room temperature has only a minor effect on the properties of the conventional C (rgdyc) -PEG-Cy5-C ' spot, thus maximizing the probability of clinical transformation.

Generalization of the PPSMI method. Can also makeSimilar methods of attaching DFO to the C (RGDyC) -PEG-C' site were used to introduce other types of functional ligands. For example, 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid (DOTA) is treated with a radioisotope (e.g., as ¹⁷⁷ Lu) one of the most effective chelators for radiolabeling. By replacing DFO-NCS with DOTA-NCS, a narrow distribution of DOTA-functionalized C (RGDyC) -PEG-C 'sites, i.e., DOTA-C (RGDyC) -PEG-C' sites, can be generated, thereby enabling, for example, the performance of ¹⁷⁷ Lu radiolabel (FIGS. 4E to H). The method is also applicable to other types of conjugation chemistry. For example, by replacing the amino-silane with a thiol-silane in a surface modification step after pegylation, thiol-functionalized C' sites can be created where additional thiol groups can be further reacted with, for example, a maleimido-functionalized ligand by, for example, a thiol-ene click reaction. To demonstrate, thiol-silane was introduced by this method onto the silica surface of PEG-Cy5-C 'spots, generating a narrow distribution of SH-PEG-Cy 5-C' spots (fig. 13A to C, table 1). Then, prior to the purification step, maleimide functionalized FITC dye (mal-FITC) was added to the reaction mixture at SH-PEG-Cy 5-C' to functionalize NPs with pH sensing FITC dye by conjugation of thiol-maleimide (fig. 1). The resulting FITC-PEG-Cy 5-C' spot showed a clean GPC elution pattern after purification, indicating a high quality NP product (FIG. 13D). FCS characterization indicated an average particle size of 6.7nm (fig. 13E, table 1). The purified FITC-PEG-Cy5-C 'spot showed an additional absorption characteristic at a wavelength of about 450nm, compared to the PEG-Cy 5-C' spot, which corresponds to the characteristic absorption of the FITC dye (FIG. 13F). The results show the successful attachment of the FITC dye by thiol-ene click reaction, and the accessibility of the thiol group at the thiol-PEG-Cy 5-C' site.

In addition to the above-described amino-silane/NCS-ligand and thiol-silane/mal-ligand chemistries, other types of conjugation chemistries, such as azido-silane/alkyne-ligand (see below), can also be applied using similar methods.

The PPSMI synthesis method can generate a family of C 'sites with different functionalities, including amine and thiol functionalized C' sites. These reactive C'The dots may become a useful platform to allow for subsequent modifications, i.e., the use of various ligands after NP preparation, depending on the intended application. But especially in the use of NH ₂ When working with (rgdyc) -Cy5-C, there is a disadvantage that they show reduced NP stability. For NH ₂ -C (rgdyc) -Cy 5-C', peaks associated with small molar mass species appeared in the GPC elution profile starting 6 months after NP preparation (fig. 14A). This may be due to, for example, peptide groups released from the NP surface. In contrast, the conventional C (rgdyc) -PEG-C point exhibited unique NP stability over a period of at least two years, with no change in either NP characteristics or in vivo performance. Therefore, we suspect that the degradation at the amination C' site may be due to the local high pH environment around the primary amine group accelerating the silane hydrolysis.

In contrast, no substantial changes were observed in the GPC elution profile and FCS characterization at the DFO-C (rgdyc) -Cy 5-C' points 6 months after NP preparation using the amine-based PPSMI method, indicating the desired NP stability (fig. 14B). This is probably due to the capping of the primary amine group by DFO-NCS during the modification reaction after PEGylation, which shortens the lifetime of the C' site with a reactive primary amine group. For this reason, while it is in principle possible to prepare the amine and thiol functionalized C' sites separately after NP preparation for further modification, it is most desirable to react these functional groups directly by introducing other functional NP ligands during NP preparation to rapidly convert these reactive groups to achieve the desired NP stability and long product shelf life.

Tetrafunctional C' site with three types of functional surface ligands: modularization of the PPSMI method. The different conjugation chemistries described above can be combined during the surface modification reaction after pegylation, providing a modular approach to the multifunctional C' site, all within a one-pot synthesis.

For demonstration, after PEGylation, the C (RGDyC) -PEG-Cy 5-C' site was first functionalized with amine groups, thereby generating NH ₂ -C (RGDyC) -PEG-Cy 5-C' spot. The DFO-NCS chelator is then attached to these C' sites via amine-NCS conjugation, thus capping the primary amine groups. Then, the resulting DFO-C (RGDyC) -PEG-Cy 5-C' was spottedOne step functionalization with thiol-silane generated SH-DFO-C (RGDyC) -PEG-Cy 5-C' spot. Finally, the FITC-mal pH sensing dye was inserted into the PEG layer and attached to the C' point silica surface by thiol-ene click reaction, blocking the reactive thiol group (fig. 1). The absorbance measurements confirmed that the resulting narrow distribution FITC-DFO-C (RGDyC) -PEG-Cy 5-C' spot integrated a total of four functional groups (FIG. 5A), while both GPC and FCS characterization suggested that the particle size at 6.8nm remained narrow and below 7nm (FIGS. 5B and C). The number of Cy5, c (rgdyc), DFO and FITC groups for each NP was estimated using a set of absorbance criteria for each individual component according to the deconvolution method described above (fig. 15 and table 1). C the (rgdyc) peptide was attached to the outside of the C' spot PEG layer, enabling active targeting of the tumor. DFO and FITC ligands capable of radioisotope chelation and pH sensing, respectively, were inserted into the PEG layer. This design ensures that they can be contacted with small ions, but at the same time reduces the potential negative effects on the in vivo performance of the C' site. The NPs showed diverse fluorescence intensities at about 525nm at different pH values when excited at 488nm (fig. 5D). This fluorescence can be attributed to the emission of the pH sensitive FITC dye inserted into the PEG layer. In contrast, NP showed pH independent fluorescence at about 675nm when excited at 645nm (fig. 5E). This fluorescence corresponds to the Cy5 dye inside the silica core. This bright NIR fluorescence can be used not only for optical imaging using the C' spot (e.g., for image-guided surgery), but also as a reference signal for ratio-type pH sensing. To this end, a ratio-type calibration curve was developed by dividing the peak sensor emission intensity (525nm) by the peak reference emission intensity (660nm) as a function of pH (FIG. 5F).

Interestingly, compared to the previously reported pH sensing C-point (i.e., from pH 4.5 to pH 8, with an intermediate pH of about 6.5) ⁴¹ The pH response of the FITC-DFO-C (RGDyC) -PEG-Cy 5-C' spot covers a broader pH range (FIG. 5E) (i.e., from pH 4 to pH 10, with an intermediate pH of about 7). This is probably caused by the blocked secondary amine groups underneath the PEG layer at the site FITC-DFO-C (RGDyC) -PEG-Cy 5-C', which affects the local acidity around the FITC dye. The co-localization of FITC and amine groups in the PEG layer, in addition to other particle functions, confers unique pH sensitivity to the C' point. Such design strategy is also providedA concept was provided to further tune the optical properties of the C' point by carefully tuning the chemical environment around the dye (i.e., the silica core and PEG layer).

Orthogonality of the ppmi method. One of the most significant advantages of the above-described PPSMI process is that it is supported by a rich kit of sophisticated, modular and orthogonal conjugation chemistries. For example, in addition to the above-described amino-silane/NCS-ligand and thiol-silane/mal-ligand chemistries, other types of conjugation chemistries, e.g., azido-silane/alkyne-ligand, can also be applied using similar methods. Furthermore, since these conjugation reactions using different chemistries do not generally interfere with each other, different functional ligands can be attached to the C' site simultaneously.

For demonstration, in contrast to the sequential addition of amino-silane, DFO-NCS, thiol-silane and FITC-mal, amino-silane and thiol-silane were added together to the C (RGDyC) -PEG-Cy5-C 'site reaction to functionalize the C' site with both amino and thiol groups. DFO-NCS and FITC-mal were then added together to convert both amine and thiol groups to functional ligands. After purification, the resulting spots FITC-DFO-C (RGDyC) -Cy 5-C' showed the desired GPC elution pattern, and each FCS characterization had an average diameter of about 6.7nm (FIGS. 16A and B). The absorption of each individual fraction was deconvoluted by fitting UV-vis spectra of the purified particles (fig. 16C), including Cy5, C (rgdyc), DFO and FITC (fig. 16D to G). This indicates that surface modification was successfully performed by simultaneously performing conjugation reaction with DFO and FITC. The PPSMI method is a powerful method of functionalizing pegylated silica NPs because the orthogonality of conjugation using PPSMI significantly shortens the reaction time required for surface modification, thereby further simplifying the preparation of multifunctional silica NPs.

Pentafunctional C' sites with four types of functional surface ligands were obtained by performing a combination of surface modifications during and after pegylation. We have described that different types of functional groups can be introduced to the C' spot surface either during the pegylation step or by surface modification after pegylation. The combination of these two approaches provides a modular and orthogonal approach to maximize the number of surface functional groups at the multifunctional C' site, all within a one-pot synthesis in aqueous media. To this end, for demonstration, we synthesized a pentafunctional C' spot with a total of four different functional ligands on the NP surface (fig. 1), allowing simultaneous optical tracing, specific cell targeting, ratiometric sensing, radioactive metal chelation and potential drug delivery.

To synthesize the pentafunctional C' site, the NP surface was first modified with specific cell targeting groups (i.e., C (rgdyc) peptide) and maleimide groups in a pegylation step by co-condensing C (rgdyc) -PEG-silane, mal-PEG-silane, and PEG-silane onto the surface of the silica core as described above. After pegylation, a thiol-modified drug small molecule is added to the reaction mixture to attach (thereby terminate) the maleimide group on the surface of the C' dot by a thiol-ene click reaction. For demonstration, we chose efavirenz (EFV, see figure 6A for molecular structure) as an example of a small therapeutic drug, a well-known antiretroviral drug for the treatment and prevention of HIV/AIDS. EFVs contain an alkynyl group that is reacted in a separate step with a heterobifunctional azido-PEG-thiol by click chemistry to modify the EFV with a thiol group (fig. 17A). In addition, EFV exhibits unique absorption characteristics in the wavelength range below 300nm, unlike the absorption of other functional groups on the pentafunctional C' point (fig. 17B and C). It should be noted that other drug linker conjugation strategies may be used herein, including the enzyme-cleavable drug-linker conjugates we previously described. After attachment of thiol-modified EFV to the C (rgdyc) -mal-PEG-Cy5-C ' spot, amino-silane, DFO-NCS, thiol-silane, and FITC-mal were added to the reaction mixture in the order following the PPSMI synthesis method for the tetrafunctional C ' spot described above to further introduce DFO and FITC groups to the EFV-C (rgdyc) -PEG-Cy5-C ' spot to achieve radioactive metal chelation and ratiometric pH sensing, respectively.

The resulting spot FITC-DFO-EFV-C (RGDyC) -PEG-Cy5-C showed the desired GPC elution profile after purification, indicating a higher particle purity (FIG. 6A). The FCS autocorrelation curve was well fitted using a single mode correlation function, indicating a narrow particle size distribution (fig. 6B). The absorbance at the FITC-DFO-EFV-C (RGDyC) -PEG-Cy 5-C' spot showed multiple peaks throughout the spectrum, which could be successfully deconvoluted to the independent contribution of each component by fitting the spectrum using a set of absorption spectrum standards (FIGS. 6D to H). The specific average number of each functional group at the C' point was then estimated by dividing the concentration of each component by the concentration of NP obtained from FCS (table 1 and fig. 6C to H), in a manner similar to that described above. Although a total of four different types of functional groups were attached to the surface of the C 'spot, the average particle size was 6.9nm, still below 7nm, still very close to the size of the clinically transformed C' spot (Table 1).

In this example, we describe a one-pot synthesis method for introducing multiple (here: up to four) types of functional ligands to fluorescent ultrasmall (diameter) in a modular and partially orthogonal manner<10nm) of pegylated silica-based nanoparticles (here, the C' dots). Different types of functional groups can be introduced in the pegylation step by co-condensing different heterobifunctional PEG-silanes onto the surface of the silica core. However, we have demonstrated that this and similar methods involving the introduction of functional groups in the pegylation step suffer from some limitations and often lead to the generation of undesirable characteristics, such as particle aggregation. These limitations can be overcome by reactions that insert-modify the Pegylated Posterior Surface (PPSMI). After pegylation, small and orthogonally reactive ligands (e.g., amine-and/or thiol-silane molecules) can be inserted into the PEG layer and attached to the silica surface by silane condensation, thereby achieving modular NP modification with functionality beyond fluorescent and tumor targeting ligands used to track particles. For example, for a device such as, for example, ⁸⁹ zr and ¹⁷⁷ lu labels, some of the most effective chelators DFO and DOTA, can be attached to the silica surface between PEG chains and at the C' point by reaction with amine or thiol groups, respectively. The PPSMI method of functionalizing C' dot-type pegylated silica nanoparticles can be generalized by employing other types of widely used conjugation chemistries, including thiol-ene and azido-alkyne click reactions. In addition, the post-surface insertion by pegylation with different conjugation chemistries can be performedThe modifications combine to provide multifunctional C' points in a modular and orthogonal manner. Finally, to maximize surface functionality, post-pegylation surface modification by inserting ligands between PEG chains can be combined with surface modification performed during the pegylation step, thereby creating surface ligands outside the surface to which the PEG chains are attached. To demonstrate, we synthesized pentafunctional DFO-FITC-EFV-C (rgbyc) -PEG-Cy 5-C' spots, which bound a total of five types of ligand groups/functionalities on a single particle: NIR fluorescent dyes in the core of particles for optical particle imaging, peptide ligands for specific tumor cell targeting, pH sensing dyes for quantitative ratiometric sensing, radioactive metal chelators for Positron Emission Tomography (PET) imaging and radiotherapy, and ligand-drug conjugates that render the particles therapeutically diagnostic and disease-treating capable. All this is achieved while also maintaining the properties of the nanoparticles, such as ultra small particle size below 7nm, higher pegylation density, good colloidal stability and control of the individual ligand numbers/surface densities, which should maintain good biodistribution and PK properties required for particle conversion into the clinic. Although there are a number of specific C 'points that have been approved by the FDA for use in human clinical trials as IND, this work opens the door for further diversification of the C' point clinical application.

Chemicals and reagents. All materials were used as received. Dimethyl sulfoxide (DMSO), isopropanol, (3-mercaptopropyl) trimethoxysilane (MPTMS), (3-aminopropyl) triethoxysilane (APTES), (3-aminopropyl) trimethoxysilane (APTMS), tetramethyl orthosilicate (TMOS), Efavirenz (EFV) and 2.0M ammonia in ethanol were purchased from Sigma Aldrich. Methoxy-terminated poly (ethylene glycol) chains (PEG-silane, molar mass about 500) were purchased from Gelest. Heterobifunctional PEG with maleimide and NHS ester groups (mal-PEG-NHS, molar mass about 870) was purchased from Quanta BioDesign. The hetero-functional azido-PEG-thiol (molar mass about 600) was purchased from Nanocs Inc. Cy5 and Cy5.5 fluorescent dyes were purchased from GE, and CW800 fluorescent dye was purchased from Li-cor. S-2- (4-Benzysothiocyanato-benzyl) -1,4,7, 10-tetraazacyclododecane tetraacetic acid (DOTA-NCS) and 1- (4-phenylisothiocyanate) -3- [6, 17-dihydroxy-7, 10,18, 21-tetraoxo-27- (N-acetylhydroxyamino) -6,11,17, 22-tetraazaheptane-eicosatetraenoic (tetraazaheptaenoic) ] thiourea (DFO-NCS) were purchased from Macrocyclics. The cyclic (arginine-glycine-aspartic acid-D-tyrosine-cysteine) Peptide (c (rgbyc)) was purchased from Peptide International. Deionized water (DI water) was generated using a Millipore Milli-Q system.

General nomenclature used to describe the chemistry of different C' points. The nomenclature presented herein and used hereinafter to distinguish between the different C' point chemistries is shown in FIG. 7 (see also Table 1). Specifically, the first term from left denotes NP product platforms, e.g., water-based C' dots or single pore mesoporous silica nanoparticles, mC dots. ³⁴ The specific dye covalently encapsulated in the silica matrix is given in parentheses behind the NP platform (e.g., Cy5 in fig. 7). The contents on the right side of the parenthesis describe the components attached to the NP surface in order of attachment (since the dots are synthesized first, they appear on the far left, not on the right as in the current abbreviated name). To highlight the relevance of functional groups on the particle surface, we introduce the following differences: a component appearing to the right of the dashed line indicates that it is directly attached to the component to the left of the dashed line. Conversely, if a component appears to the right of the underline, that component is directly attached to the surface of the silica NP, rather than to the preceding component to the left of the underline. In contrast to the groups used for conjugation chemistry (e.g., mal-thiol, which describes conjugation between maleimide and thiol groups), components that define function, e.g., targeting ligands or other fluorescent dyes, are indicated in bold and italics (see fig. 7 and table 1). The number of ethylene glycol monomer units is shown if the functional group is attached to the silica NP surface through a PEG chain, such as PEG6 or PEG 12. As mentioned in the introduction, the name of a nanoparticle carries in this way accurate information about: (i) an NP platform; (ii) an encapsulated fluorescent dye; (iii) specific surface functionality and its connectivity to the particles; (iv) (iv) a specific conjugation chemistry, (v) a synthetic sequence, or (vi) a specific PEG chain length.

Synthesis of C' Point (dye) -PEG 12-mal-thiol-C (RGDyC) PEG6 functionalized with different types of NIR dyes. Detailed synthesis of C (rgdyc) functionalized C' site is described in our previous publication. To encapsulate different types of NIR dyes, silane-functionalized dyes (e.g., Cy 5-silane, cy5.5-silane, and CW 800-silane) are added to the reaction mixture along with TMOS in the first step of the point C synthesis. The remaining NP synthesis was identical to the previously reported protocol for C (rgdyc) functionalized C' sites.

Synthesis of C' Point (dye) -PEG 12-mal-thiol-C (RGDyC) A PEG6 with an increased number of C (RGDyC) per particle. The concentration of C (RGDyC) -PEG-silane was raised from the initial 0.69mM (which generally resulted in a C (RGDyC) peptide number between 16 and 25 per C' spot) to 1.73mM to further increase the amount of C (RGDyC) per NP. To prevent NP aggregation, the reaction concentration of ammonium hydroxide was also increased from 2mM to6 mM. The method of achieving this may be to start the C' point synthesis at a concentration of 6mM ammonium hydroxide, or to add additional ammonium hydroxide immediately prior to the PEGylation step to raise the concentration of ammonium hydroxide to6 mM. Only slight differences in the final C' point products produced by these two slightly different methods were observed. Therefore, in order to simplify the preparation process, it is preferred to start the synthesis at an ammonium concentration of 6 mM.

Synthesis of C ' Point (dye) -PEG 12-mal-thiol-C (RGDyC) -PEG 12-mal-PEG 6, C ' Point (dye) -PEG 12-mal-thiol-C (RGDyC) -amido-NCS-DFO _ PEG6, and C ' Point (dye) -PEG 12-mal-thiol-C (RGDyC) -PEG 4-DBCO _ PEG 6. To introduce additional functional ligands to the C' site (dye) -PEG 12-mal-thiol-C (rgdyc) -PEG6, the ligand-silane conjugate was added to the reaction mixture along with C (rgdyc) -PEG-silane and PEG-silane in the pegylation step. These ligand-silane conjugates include maleimide-functionalized heterobifunctional PEG-silanes (mal-PEG-silane), DFO-functionalized silanes (DFO-NCS-amino-silane), and DBCO-functionalized heterobifunctional PEG-silanes (DBCO-PEG-silane). The concentration of these ligand-silane conjugates ranges from 0.01mM to 0.34mM, depending on the requirements on the surface ligand density. The C' site (dye) -PEG 12-mal-thiol-C (rggyc) -PEG 12-mal-PEG 6 produced by this method showed desirable reactivity to thiol-functionalized ligands. However, C 'point (dye) -PEG 12-mal-thiol-C (RGDyC) amidoamine-NCS-DFO-PEG 6 and C' point (dye) -PEG 12-mal-thiol-C (RGDyC) PEG 4-DBCO-PEG 6 exhibit an undesirable profile or non-uniform distribution of ligands.

Synthesis of C 'point (dye) -amine-PEG 12-mal-thiol-C (RGDyC) -PEG6 and C' point (dye) -PEG 12-mal-thiol-C (RGDyC) -PEG 6-amine. To introduce amine groups at the C 'site, an amine-silane (i.e., APTMS) was introduced into the reaction mixture at a different step of the C' site synthesis. In one way, APTMS was added to the reaction mixture at a concentration of 1.7mM with vigorous stirring, followed by the addition of c (RGDyC) -PEG-silane and PEG-silane, the remainder of the synthesis remaining unchanged. The resulting C' dot (dye) -amine-PEG 12-mal-thiol-C (RGDyC) -PEG6 showed a broader NP size distribution. At the same time, degradation of Cy5 dye was observed. In the post-pegylation method, APTMS is added after the pegylation step. This was done by first reducing the reaction temperature from 80 ℃ to room temperature at the end of the pegylation step and then adding APTMS at a concentration of 2.3mM with vigorous stirring. The reaction mixture was then left overnight at room temperature with vigorous stirring and then purified. GPC and UV-vis characterization showed that the resulting C' dot (dye) -PEG 12-mal-thiol-C (rgbyc) -PEG6_ amine exhibited the desired NP size distribution and good absorption characteristics. In this way, the C' site (dye) -PEG 12-mal-thiol-C (RGDyC) -PEG 6-amine can be successfully prepared at APTMS concentrations as low as 0.3mM without the NP product losing reactivity with amine reactive ligands.

PPSMI-based synthesis of C 'site (dye) -PEG 12-mal-thiol-C (RGDyC) PEG6_ amino-NCS-DFO and C' site (dye) -PEG 12-mal-thiol-C (RGDyC) PEG6_ amino-NCS-DOTA. The C ' site (dye) -PEG 12-mal-thiol-C (RGDyC) -PEG6_ amino-NCS-DFO and C ' site (dye) -PEG 12-mal-thiol-C (RGDyC) -PEG6_ amino-NCS-DOTA were synthesized by adding DFO-NCS or DOTA-NCS to a reaction mixture of C ' site (dye) -PEG 12-mal-thiol-C (RGDyC) -PEG6_ amine, respectively, one day after the addition of amino-silane, at room temperature with vigorous stirring. The reaction was left overnight at room temperature with vigorous stirring before purification. The concentrations of DFO-NCS and DOTA-NCS were varied between 0.02mM and 0.4mM to vary the amount of DFO or DBCO per NP. Both the concentrations of APTMS and DFO were varied to control the amount of DFO per NP.

PPSMI-based synthesis of C 'site (dye) -PEG6_ thiol and C' site (dye) -PEG6_ thiol-mal-FITC. To introduce a thiol group into the PEG-C ' site to generate a thiol-functionalized C ' site (dye) -PEG6_ thiol, the synthesis of C ' site (dye) -PEG 12-mal-thiol-C (rgbyc) -PEG6_ amine was adjusted by substituting thiol-silane for amino-silane and using C ' site (dye) -PEG6 as the base NP instead of C ' site (dye) -PEG 12-mal-thiol-C (rgbyc) -PEG 6. The remainder of the NP synthesis was identical to that of the C' -point (dye) -PEG 12-mal-thiol-C (RGDyC) -PEG 6-amine. To further attach the FITC dye to the C ' point (dye) -PEG6_ thiol to generate C ' point (dye) -PEG6_ thiol-mal-FITC, the synthesis of C ' point (dye) -PEG 12-mal-thiol-C (rggyc) _ PEG6_ amino-NCS-DFO was adjusted by substituting DFO-NCS with FITC-mal and using C ' point (dye) -PEG6_ thiol as the base NP instead of C ' point (dye) -PEG 12-mal-thiol-C (rggyc) _ PEG 6-amine. The remainder of the NP synthesis was identical to that of C' -site (dye) -PEG 12-mal-thiol-C (RGDyC) -PEG6_ amino-NCS-DFO.

PPSMI-based synthesis of tetrafunctional C' dots (Cy5) -PEG 12-mal-thiol-C (RGDyC) -PEG6_ amino-NCS-DFO _ thiol-mal-FITC. C ' dots (Cy5) -PEG 12-mal-thiol-C (RGDyC) -PEG6_ amino-NCS-DFO-mal-FITC were synthesized by first introducing a DFO group to the C ' dots (Cy5) -PEG 12-mal-thiol-C (RGDyC) -PEG6 via the described synthesis method for C ' dots (dye) -PEG 12-mal-thiol-C (RGDyC) -PEG6_ amino-NCS-DFO. Then, thiol groups were added to the C 'spot (dye) -PEG6_ thiol by the synthetic method to generate C' spot (Cy5) -PEG 12-mal-thiol-C (rgbyc) -PEG6_ amino-NCS-DFO _ thiol. In the last step, the FITC dye was attached to the C' point (dye) -PEG6_ thiol-mal-FITC by the synthesis method. The resulting C' dots (Cy5) -PEG 12-mal-thiol-C (RGDyC) _ PEG6_ amino-NCS-DFO _ thiol-mal-FITC were finally purified by GPC and characterized FCS and optically. The same C' spot (Cy5) -PEG 12-mal-thiol-C (rgbyc) -PEG6_ amino-NCS-DFO _ thiol-mal-FITC was also synthesized by an alternative method in which the amino-silane and thiol-silane were added to the reaction mixture together, rather than sequentially. The reaction was then left at room temperature with vigorous stirring for 4 hours, after which DFO-NCS and FITC-mal were added simultaneously. The rest of the reaction remained unchanged.

Synthesis of C' dot (Cy5) -PEG 12-mal-thiol-C (RGDyC) _ PEG 12-mal-thiol-PEG 10-EVF _ PEG6_ amino-NCS-DFO _ thiol-mal-FITC. The C' dots (Cy5) -PEG 12-mal-thiol-C (rgbyc) PEG 12-mal-thiol-PEG 10-EVF _ PEG6_ amino-NCS-DFO _ thiol-mal-FITC were first synthesized by conjugating EFV to azido-PEG-thiol in DMSO at a molar ratio of 4EFV:1 azido-PEG-thiol (EFV concentration of about 0.021M) via azido-alkyne click chemistry. The reaction mixture was left under nitrogen for 6 days to achieve the desired conjugation yield. The resulting thiol-functionalized EFV was then added to the reaction mixture of C' dot (Cy5) -PEG 12-mal-thiol-C (rgbyc) -PEG12-mal _ PEG6 at a concentration of 1.15mM after the pegylation step (i.e., after the 80 ℃ heat treatment step). The reaction mixture was then allowed to stand at room temperature with vigorous stirring for an additional 6 days to attach thiol-functionalized EFV to the maleimide group on C 'spot (Cy5) -PEG 12-mal-thiol-C (rgdyc) -PEG 12-mal-PEG 6, yielding C' spot (Cy5) -PEG 12-mal-thiol-C (rgdyc) -PEG 12-mal-thiol-PEG 10-EVF _ PEG 6. Subsequently, DFO and FITC were introduced following the same procedure as described above for C' dot (Cy5) -PEG 12-mal-thiol-C (RGDyC) _ PEG6_ amino-NCS-DFO _ thiol-mal-FITC.

C GPC characterization and purification of (RGDyC) -PEG-C' dots. The detailed GPC characterization and purification steps at point C' have been described in our previous publication.

UV-vis and FCS measurements. The absorption spectra of the samples were measured on a Varian Cary 5000 spectrophotometer. FCS measurements were performed using the homemade FCS/FCCS setup, as described previously. Cy5 and Cy5.5 dots were excited using a 635nm solid state laser, and cw800 dots were excited using a 785nm solid state laser.

General nomenclature used to describe the chemistry of different C' points. The nomenclature presented herein and used hereinafter to distinguish between the different C' point chemistries is shown in FIG. 7 (see also Table 1). Specifically, the first term from left denotes NP product platforms, e.g., water-based C' dots or single pore mesoporous silica nanoparticles, mC dots. The specific dye covalently encapsulated in the silica matrix is given in parentheses behind the NP platform (e.g., Cy5 in fig. 7). The contents on the right of the parenthesis describe the components attached to the NP surface in order of attachment (since the dots are synthesized first, they appear to the far left, not to the right as we have the name at hand). To highlight the relevance of functional groups on the particle surface, we introduce the following differences: a component appearing to the right of the dashed line indicates that it is directly attached to the component to the left of the dashed line. Conversely, if a component appears to the right of the underline, that component is directly attached to the surface of the silica NP, rather than to the preceding component to the left of the underline. In contrast to the groups used for conjugation chemistry (e.g., mal-thiol, which describes conjugation between maleimide and thiol groups), components that define function, e.g., targeting ligands or other fluorescent dyes, are indicated in bold and italics (see fig. 7 and table 1). The number of ethylene glycol monomer units is shown if the functional group is attached to the silica NP surface through a PEG chain, such as PEG6 or PEG 12. As mentioned in the introduction, the name of a nanoparticle carries in this way accurate information about: (i) an NP platform; (ii) an encapsulated fluorescent dye; (iii) specific surface functionality and its connectivity to the particles; (iv) (iv) specific conjugation chemistry, (v) synthetic sequence, or (vi) specific PEG chain length.

Example 2

This example provides examples of the methods and compositions of the present disclosure and uses thereof.

An example of a functional C 'spot synthesized by the PPSMI method, namely the DBCO-PEG-Cy 5-C' spot, is described (FIG. 18). The DBCO-PEG-Cy5-C 'dot contains DBCO-reactive groups between the PEG chains on the surface of the C' dot. The DBCO group can be further reacted with the azido-conjugated functional ligand by click chemistry, thereby introducing additional functional groups to the C' site. To generate DBCO-PEG-Cy5-C 'dots, silane-conjugated Cy5 fluorescent dye was first covalently encapsulated in the silica nanoparticles during formation of the C' dot silica core. PEG-silane was then covalently attached to the surface of the silica nanoparticles, forming pegylated PEG-Cy 5-C' spots. After the pegylation step, an amino-silane is added to the reaction mixture to introduce reactive amine groups by silane condensation covalently attached to the remaining silanol groups under the C' point PEG layer. DBCO-PEG4-NHS ester was then added to further react with amine groups under the C' point PEG layer via an amine-NHS ester conjugation reaction. Varying concentrations of DBCO-PEG4-NHS ester were used to alter the number of DBCO ligands at the DBCO-PEG-Cy 5-C' spot.

Example 3

This example provides examples of the methods and compositions of the present disclosure and uses thereof.

An example of a functional C 'spot synthesized by the PPSMI method, i.e., a DBCO-DFO-PEG-Cy 5-C' spot, is described (FIG. 19). The DBCO-DFO-PEG-Cy5-C 'site contains DBCO-reactive groups and DFO chelating groups between PEG chains on the surface of the C' site. Although the DBCO group can be further reacted with the azido-conjugated functional ligand by click chemistry to introduce additional functional groups to the C 'site, the DFO group can chelate radioactive metals, thereby making the C' site useful as a probe for Positron Emission Tomography (PET). To generate DBCO-DFO-PEG-Cy5-C 'dots, a silane-conjugated Cy5 fluorescent dye was first covalently encapsulated in a silica nanoparticle during formation of the C' dot silica core. PEG-silane was then covalently attached to the surface of the silica nanoparticles, forming pegylated PEG-Cy 5-C' spots. After the pegylation step, an amino-silane is added to the reaction mixture to introduce reactive amine groups by silane condensation covalently attached to the remaining silanol groups under the C' point PEG layer. SCN functionalized DFO (DFO-SCN) was then added to the reaction mixture to react with the amine groups under the C' dot PEG layer by an amine-SCN conjugation reaction. Low concentrations of DFO-SCN were used to introduce less than 5 DFO chelators per C' point. Thus, as described above, the remaining amine groups on the surface of the C' dot are available for DBCO attachment. One day after addition of DFO-SCN, DBCO-PEG4-NHS ester was then added to the reaction mixture to further react with the remaining amine groups under the C' dot PEG layer via an amine-NHS ester conjugation reaction. As a result, both DFO and DBCO were covalently attached to the surface of the C' point between PEG chains by the PPSMI method.

Example 4

This example provides a summary of the reaction conditions described in this disclosure.

The synthesis system can be extended to at least nine orthogonal and modular functionalization pathways to selectively combine different functional groups onto a single nanoparticle platform. Nanoparticle platforms to which these functionalization methods are applicable include, but are not limited to, C 'sites, AlC' sites, and mC sites. (in Table 2 ¹ )

In addition to Cy5, Cy5.5, and cw800, other types of dyes can also be functionalized with silane groups by conjugation chemistries including, but not limited to, thiol-ene, amine-NHS ester, azido-alkyne chemistry. Dye-silane conjugates can then be added during the core formation step to impart different fluorescent properties to the nanoparticles. Different types of dyes include, but are not limited to, DACM, DEAC, RHG, TMR, TRITC, FITC, Cy3, ATTO647N, ATTO680, and DY 782. (in Table 2 ² )

In addition to c (rgbyc) cancer targeting peptides, other ligands with application-specific functions can also be attached to heterobifunctional PEG-silanes by conjugation chemistries including, but not limited to, thiol-ene, amine-NHS ester, azido-alkyne chemistry. The resulting ligand-PEG-silane conjugate can then be added in a pegylation step to impart different functionalities to the nanoparticle. Different functional ligands include, but are not limited to, peptides, antibody fragments, DNA molecules, RNA molecules, fluorescent dyes, sensor molecules, drug molecules, and chelator molecules. (3 in Table 2)

In addition to maleimide functionalized heterobifunctional PEG-silanes, other heterobifunctional PEG-silanes containing reactive groups may be attached to the nanoparticle surface during the pegylation step, allowing further modification of the nanoparticle with additional functional ligands at the end of the PEG chain. Reactive groups include, but are not limited to, maleimide groups, NHS ester groups, azide groups, amine groups, thiol groups, alkynyl groups, and DBCO groups. (in Table 2 ⁴ )

In addition to EFV-PEG-SH, following PEGylation, can be made specific by conjugation chemistryOther ligands that apply functionality are attached to reactive groups already present at the end of the PEG chain on the surface of the nanoparticle, including but not limited to thiol-ene reactions, amine-NHS ester reactions or azide-alkyne reactions. Different functional ligands include, but are not limited to, peptides, antibody fragments, DNA molecules, RNA molecules, fluorescent dyes, sensor molecules, drug molecules, and chelator molecules. (in Table 2 ⁵ )

In addition to thiol-and amino-silanes, other silane conjugates containing reactive groups can be inserted into the PEG layer after pegylation and attached to the underlying silica surface, allowing further modification of the nanoparticle between PEG chains with additional functional ligands. Reactive groups include, but are not limited to, maleimide groups, NHS ester groups, azide groups, amine groups, thiol groups, alkynyl groups, and DBCO groups. (in Table 2 ⁶ )

In addition to DFO, DOTA and FITC, other ligands with application specific functions can be attached to reactive groups that have been inserted into the PEG layer after pegylation. Different functional ligands include, but are not limited to, peptides, antibody fragments, DNA molecules, RNA molecules, fluorescent dyes, sensor molecules, and chelator molecules. (in Table 2 ⁷ )

Other silane conjugates containing small ligands with specific application functions can be inserted into the PEG layer after pegylation and attached to the underlying silica surface. Different functional ligands include, but are not limited to, peptides, antibody fragments, DNA molecules, RNA molecules, fluorescent dyes, sensor molecules, and chelator molecules. (in Table 2 ⁸ )

Claims (14)

1. A method of forming functionalized polyethylene glycol (PEG) functionalized silica nanoparticles, the method comprising:

a step of modifying a pegylated rear surface (PPSMI) by insertion, wherein the PPSMI step comprises covalently inserting a silane having orthogonal functional groups between PEG chains of the nanoparticle and on the silica surface, and wherein the method comprises using an aqueous reaction medium.

2. The method of claim 1, wherein the nanoparticle is a pegylated core or core-shell silica nanoparticle.

3. The method of claim 1, wherein the nanoparticle is a pegylated core or core-shell silica nanoparticle less than 10 nm.

4. The method of claim 1, comprising modularly and orthogonally functionalizing the pegylated silica nanoparticles after the PPSMI step with from 1 to 5 types of different functional ligands on the nanoparticle surface.

5. The method of claim 1, comprising modularly and orthogonally functionalizing pegylated silica nanoparticles after the PPSMI step with 2 types of different functional ligands on the nanoparticle surface.

6. The method of claim 1, comprising modularly and orthogonally functionalizing pegylated silica nanoparticles after the PPSMI step with 3, 4 or 5 types of different functional ligands on the nanoparticle surface.

7. The method of any one of claims 4-6, wherein the functional ligand comprises a reactive group selected from the group consisting of: amine groups, thiol groups, NHS ester groups, maleimide groups, alkynyl groups, azide groups, and DBCO groups.

8. The process of claim 1 wherein the aqueous reaction medium is free of organic solvents other than 10% or greater of polar aprotic solvent.

9. The process of claim 1, wherein the aqueous reaction medium does not contain 5% or more alcohol.

10. The method of claim 1, comprising adjusting the pH of the aqueous reaction mixture to a desired value or within a desired range.

11. The method of claim 10, wherein adjusting the pH of the aqueous reaction mixture comprises adding a base.

12. The method of claim 11, wherein the base is ammonium hydroxide.

13. The method of claim 1, wherein the nanoparticles comprise one or more fluorescent dye molecules encapsulated therein.

14. The process of claim 1, wherein the process involving the PPSMI step occurs between nanoparticle pegylation and purification steps.