Silica NPs can be easily prepared on large scale with discrete, monodisperse particle sizes through the condensation reaction of tetraethyl orthosilicate (TEOS) or tetramethyl orthosilicate (TMOS). For example, monodisperse silica spheres with controlled sizes (50 nm - 2 µm) can be prepared in a reaction mixture of water, alcoholic solvent, ammonia, and alkyl silicate ester by controlling alcoholic solvents, different alkyl silicate esters, as well as the concentration of each component.28
Silane coupling agents containing a trialkoxysilane group can be readily incorporated into silica NPs during such condensation reaction.29
We reasoned that trialkoxysilane-containing drugs (dyes) through a degradable ester linker should be able to be condensed with TEOS or TMOS to allow the drug (dye) molecules to be incorporated into the resulting silica NPs, which can be released through the cleavage of the ester linker. To demonstrate this concept, we started with 1
, a trimethyl orthosilicate that contains pyrenemethanol (Pyr-OH) as the model drug. By carefully controlling reaction conditions, we were able to prepare Pyr-NCs with discrete sizes 15 nm apart between 20 nm and 80 nm. As shown in and (entry 1–5), NCs with sizes of 22.2 ± 1.7 nm (Pyr20), 36.3 ± 2.9 nm (Pyr35), 49.3 ± 2.9 nm (Pyr50), 64.1 ± 3.1 nm (Pyr65) and 80 nm (Pyr80) can be readily prepared in multigram quantities. To test the reproducibility of these conditions to prepare NCs with the corresponding size, we repeated each experiment 3 to 5 times and found that the NCs with the desired size could be precisely produced each time. For instance, the five experiment for making Pyr20 under the same condition resulted in particles with size of 22.2 ± 1.7 nm, 22.7 ± 2.4 nm, 22.9 ± 1.9 nm, 22.2 ± 1.1 nm and 26.2 ± 2.4 nm (). The CV values of these five experiments are 7.7, 10.6, 8.3, 5.0 and 9.2, respectively, with an average of CV value of 8.1%. The low CV values (< 10%) of Pyr20 NC indicate these particles are technically monodisperse by industry standard.30
The conditions of making pyrene-containing NCs of 35, 50, 65 and 80 nm showed similar control over NC size, monodispersity and reproducibility (). The hydrodynamic sizes of these Pyr-NCs were also measured by dynamic light scattering (DLS) (Supplementary Table S3
), which are larger than the hard core sizes measured by scanning electron microscopy (SEM). All the PDI values measured by DLS are below or around 0.1 indicating again the high monodispersity of these Pyr-NCs. To be consistent, all NC (NP) sizes and size distributions reported in the following part of this paper, except for those in
and Table S3
, were determined based on the SEM data of the particles, by averaging the particle size of a representative SEM image containing at least 100 particles.
Figure 1 Precise size control of drug/dye-silica nanoconjugates (NCs). (a) Preparation of pyrene-silica nanoconjugates (Pyr-NCs) with discrete size ranging from 20 to 200 nm. Three to five separate batches of Pyr-NCs for each size were prepared to demonstrate (more ...)
Preparation of drug-/dye-silica nanoconjugates[a].
Figure 4 Tunable drug release profiles and solid-form formulation of drug-silica nanoconjugates. (a) Release kinetics of Cpt-NCs with different linkers and sizes in 50% human serum at 37 °C. (b) NC size distributions measured by dynamic light scattering (more ...)
Lyophilization of Silica NC.
NCs with particle size 100 nm or larger should be much easier to prepare compared to smaller particles. As expected, both 100-nm and 200-nm Pyr-NCs (Pyr100 (101.3 ± 3.6 nm) and Pyr200 (202. 6 ± 5.9 nm)) were prepared with monodisperse size distribution (CV < 10%) and high reproducibility ( and entries 6–7 in ). Statistical significances (p
< 0.01) were found for all NCs with adjacent sizes between 20 and 200 nm. We compared the silica NCs with polymeric NPs prepared through nanoprecipitation (NPP) of amphiphilic copolymers and demonstrated the difference between these two methods for particle size control. Silica NCs can be easily prepared with monodisperse size distributions (CV < 10%). However, polymeric NPs prepared through NPP methods have polydisperse size distribution. For instance, poly(lactide-co-glycolide)-b
-methoxy-PEG (PLGA-PEG) di-block copolymer with a 13-kDa PLGA block and 5-kDa PEG, was precipitated to form PLGA-PEG NPs. The resulting NPs showed a much broader size distribution as compared to silica NCs (CV = 39.2%, entry 8, ; Supplementary Figure S1
We next attempted to incorporate therapeutics and dyes to silica NCs using the same reactions under similar conditions. Camptothecin (Cpt), a cytotoxic chemotherapeutic agent which inhibits the DNA enzyme topoisomerase I, was converted the corresponding silane derivative 2
() with Cpt connected a trialkoxysilane group via
a hydrolyzable thioether ester linker, and then incorporated into the silica NCs under similar conditions used for preparing Pyr-NCs with similar sizes. As expected, remarkable control over particle size was observed for the reaction, which resulted in monodisperse Cpt-NCs in all corresponding size ranges (entries 9–12, ). For the Cpt-NCs with expected size of 20, 50, 100 and 200 nm, the obtained NC sizes were 26.3 ± 2.5 nm, 51.5 ± 3.8 nm, 96.1 ± 8.8 nm and 222.7 ± 16.5 nm (; Supplementary Figure S2
). We also attempted to incorporate other therapeutic agents (e.g.
, paclitaxel (Ptxl)) or fluorescent dyes (e.g.
, rhodamine B (RITC) and IR783) using similar approach with corresponding silane reagents (3–5
) to prepare 20-nm and 50-nm NCs (). As expected, all obtained NCs containing Ptxl, RITC or IR783 were monodisperse (CV < 10%) and had the expected particles size (entries 13–19, ). In order to increase the systemic circulation half-life and reduce aggregation of NCs in blood,31
the surface of NCs was modified with PEG via
the use of 1-(2-(2-methoxyethoxy)ethyl)-3-(3-(trimethoxysilyl)propyl)urea (mPEG5k
(). The resulting pegylated NCs displayed remarkable stability in both PBS (1×) and cell medium containing 10% fetal bovine serum (FBS) (Supplementary Figure S3
); the NC size remained unchanged for hours.
After we prepared drug(dye)-NCs with precisely controlled size, we next studied the size effect of these new drug delivery systems on their in vivo
biodistribution, tumor tissue penetration and cellular internalization. All silica NCs involved in the following in vitro
and in vivo
studies have identical surface properties, spherical shape and chemical structures and compositions; particle size was the only parameter changed in these studies. Pegylated silica NCs with discrete sizes of 20, 50 and 200 nm containing rhodamine B (RITC) (termed RITC20, RITC50 and RITC200, respectively) were prepared at a 4:6
ratio of 10 (entries 14–16, ; Supplementary Figure S4
). To facilitate in vivo/ex vivo
analysis of fluorescent NCs with reduced autofluorescence, we prepared pegylated NCs containing IR783, a near inferred (NIR) dye at a 5:6
ratio of 10; the resulting NIR active NCs with discrete sizes of 20, 50 and 200 nm were denoted as IR20, IR50 and IR200, respectively (entries 17–19, ; Supplementary Figure S5
In the biodistribution study of IR20, IR50 or IR200 in vivo
using C57BL/6 mice bearing subcutaneously implanted Lewis lung carcinoma (LLC), tail vein intravenous (i.v.) administration of the NCs followed by tissue harvesting 24-hour later showed that majority of NCs were accumulated in liver and spleen, few were in the respiratory and urinary systems (). The fluorescence of IR was found to have excellent tissue transmission; IR concentration can be quantitatively assessed in tissues with thickness 2 mm or less (Supplementary Figure S6
). Importantly, NCs with smaller sizes distributed and accumulated in the tumor tissue more efficiently than NCs of larger sizes (). The injected doses of NCs normalized for tumor tissue weight (I.D.%/g) were 4.18 ± 0.81, 0.98 ± 0.59 and 0.52 ± 0.05 for IR20, IR50 and IR200, respectively. A decrease in particle size by 2.5-fold from 50 nm to 20 nm resulted in an increase of NC concentration by 330% in tumor tissue (from 0.98 to 4.18, **p
< 0.01). In comparison, a decrease in particle size by 4-fold from 200 nm to 50 nm resulted in an increase of NC concentration in tumor tissue by only 88% (from 0.52 to 0.98). NC size showed significant influence on the systemic and tissue biodistribution, and this effect seems to be more profound for NCs below 50 nm in size. These results underscore the importance of studying nanomedicines with sizes less than 50 nm.
Figure 2 Size effect on biodistribution and tumor penetration. (a), (b) C57BL/6 mice bearing Lewis lung carcinoma (LLC) (size: ~5.0 mm × 6.0 mm; n=3) were injected intravenously with IR20, IR50 and IR200. Mice were euthanized and the tissues were collected, (more ...)
As the silica NCs used in our study do not have targeting ligand, the accumulation of these NCs should follow the enhanced permeation and retention (EPR) effect,32
a widely recognized passive targeting mechanism, for their accumulation and retention in the tumor tissues. While the NCs extravasate the leaky vasculatures into the tumor tissues, the capability for the NCs to diffuse away from the capillary blood vessels and vasculature should have significant effect on the retention of NCs. We went on and studied the size dependency of silica NCs diffusion/penetration in tumor tissues. We performed the tumor penetration study by incubating LLC tumors (grown in C57BL/6 mice with ~200 mg) for 48 hours in culture medium containing equal concentration of IR20, IR50 or IR200. The tumor sections (20 µm in thickness) were then analyzed by NIR fluorescence microscope. As shown in the size dependency of tumor penetration was obvious with IR20 penetrating tumor tissue with the greatest depth from the periphery of the tumors, followed by IR50 with intermediate penetration depth and IR200 with limited tumor penetration. To quantify the penetration, we defined the tumor tissue penetration depth as the distance from the periphery of the tumor to the site where the fluorescence intensity decreases by 95% as compared to the tumor periphery fluorescent intensity. The penetration depths of IR20, IR50 and IR200 were found to be 1,396 µm, 660 µm and 88 µm, respectively (). The penetration depth of IR20 is twice and sixteen times of that of IR50 and IR200, respectively. To verify the size-dependency of tumor penetration in vivo
, we intravenously administered RITC20, RITC50 and RITC200 to LLC-bearing C57BL/6 mice via
tail vein. Tumors were collected 24 hours post-injection, fixed and sectioned. After the blood vessel was stained with human Von Willebrand Factor antibody (green, FITC channel in ), the tumor tissues were then analyzed using confocal microscope to study the distribution of NCs in tumor tissues relative to the blood vessels. This study showed the effect of biodistribution and diffusion collectively. RITC20 and RITC50 significantly outperformed RITC200, and diffused away from and situated distally to the blood vessel. Comparing the representative regions of interest, the fluorescence intensity of RITC20 is 4- and 22-times greater than RITC50 and RITC200, respectively. This observation of size-dependent in vivo
penetration is consistent with the observations from the tumor penetration studies using ex vivo
model () and the size-dependent biodistribution studies ().
While the NCs diffuse into tumor tissue, whether the NCs stay in interstitial extracellular matrix or are internalized and reside inside the cells should impact the penetration depth in tumor tissue as well as the capability of retention. We thus compared the size-dependent uptake of these NCs in HeLa cells. Cellular internalization of RITC20, RITC50 or RITC200 into the HeLa cells for 30, 60, or 90 minute incubation was analyzed by fluorescence-assisted flow cytometry (FACS) to assess the kinetics of NC internalization (). We found that smaller NCs were internalized into HeLa cells faster and more efficiently than NCs with larger size, in terms of both percentage of the fluorescent cells and total accumulated mean fluorescence intensity. The number of fluorescent cells accounts for 1.4%, 6.6% and 9.2% of the total treated cells for 30-, 60- and 90-minute incubation with RITC200. These numbers were 6.6%, 37.2% and 55.2% for RITC50, and 21.9%, 49.1% and 71.0% for RITC20, respectively (). The fluorescence intensities of cells for 30-, 60- and 90-minute incubation with RITC200 were 0.18, 0.25 and 0.28, in arbitrary units of FACS. These numbers were 0.23, 1.15 and 1.52 for RITC50 and 0.73, 1.99 and 5.25 for RITC20, respectively (). The 20-nm NC was therefore internalized 18.7 times and 3.5 times more than 200-nm and 50-nm NC for a total of 90-minute incubation. Interestingly, comparing the fluorescence intensity change of the three 30-minute blocks (0–30 min, 30–60 min and 60–90 min), we found the rate of internalization of 20-nm NC (RITC20) in HeLa cells were accelerating in the first 90 minutes while the accumulation of 200-nm NC (RITC200) were evidently de-accelerating (). For RITC200, 90-minute incubation versus
30-minute incubation resulted in an increase of the number of the fluorescent cells by 660% (9.2% vs.
1.4%), but the total accumulated fluorescence intensity was only increased by 56% (0.28 vs.
0.18), suggesting that not all internalized RITC200 can be effectively retained inside of the cells and exocytosis might occur simultaneously.33
In contrast, 90-minute incubation versus
30-minute incubation of RITC20 resulted in an increase of the number of fluorescence cells by 340% (71% vs.
21%) and the total accumulated fluorescence intensity by 720% (5.25 vs.
0.73), clearly indicating that 20-nm particle can be effectively internalized and retained in the cells, and the internalization/retention process become more favorable for the duration of study. The size-dependent cell-uptake and retention was also verified by confocal microscopy study (), which demonstrated that NCs with smaller sizes were internalized and retained inside the cells more efficiently than the NCs with larger sizes.
Figure 3 NC size effect on cellular internalization. (a), (b), (c) Internalization of RITC-NCs into HeLa cells over 90 min incubation at 37 °C evaluated by the percentage of cells containing internalized NCs (a), mean fluorescence of treated cells (b) (more ...)
The 20-nm silica NC outperforms the 50-nm NC and the 200-nm NC by ~2–5 times and ~10–20 times, respectively, in terms of biodistribution, tumor tissue penetration, and internalization to cancer cells. Collectively, 20-nm NCs breach the three physiological barriers (systemic, tissue, and cellular) that are critical to drug delivery significantly better than larger particles. We are currently exploring whether the NCs containing targeting ligand will follow the same size dependency as what we observed in this study with the use of non-targeting NCs. Because particles less than 10 nm may be subject to significant renal clearance and rapid fenestration into other tissues (e.g., lymphatic system), which is undesirable for sustained circulation that is critical to NC passive targeting and tumor tissue accumulation via EPR effect, NC around 20-nm may be close to the optimal size for drug delivery application.
Silica NCs have other promising properties that are noteworthy. First, this nano-fabrication process as shown in allows the incorporation of drug (dye) molecules in high yields (up to 24%) () that are comparable to or higher than the FDA-approved drug delivery systems, such as Doxil (~10%).34
Drug burst release is a long-standing formulation challenge of nanocarriers with drug encapsulated in polymeric NPs or adsorbed in mesoporous silica NPs, which causes undesirable dose dumping, significant side effects, and reduced long-term therapeutic efficacy. Since the drug release kinetics of drug-NCs is determined by the hydrolysis of the thioether ester bond linker, the release kinetics of drug from NCs are more controllable with essentially no burst release (). In human serum, Cpt20 with the hydrophobic thioether ester linker between Cpt and the silica particles showed sustained drug release with 14.8% of CPT being released in 48 hours (); the IC50
value of Cpt20 in HeLa cells was found to be 220 nM. When the linker was changed to a hydrophilic amine ester as in Cpt-N20 (entry 20, ; ), which was prepared by using 7
as the corresponding drug-containing silane reagent (), the Cpt release kinetics can be dramatically accelerated with Cpt being 100% released within 48 hours, resulting in a much lower IC50
value (9.0 nM, Supplementary Table S4
). This could be due to the fact that hydrophilic amine ester is more assessable by water and esterase which can accelerate the cleavage of the ester bond. By controlling the feed ratio of 2/7
during Cpt-NC fabrication to the ratios of these two different linkers, the Cpt release half-life can be precisely adjusted ranging from 24 hours to about two weeks.
Besides controlled particle size, drug loading and release kinetics, other issues critical to the clinical translation of NP drug delivery system, such as scalability, lyophilizability, and toxicity, should also be addressed. These issues may also present the bottleneck to the clinical translation of a nanomedicine. We found the silane chemistry could be easily used for the large-scale preparation of drug-containing NCs. We tested the preparation of one gram of 50-nm Cpt-NC in one pot, and successfully obtained NCs with the expected size (46.4 ± 4.6 nm) in quantitative yield within one day (entry 22, ; Supplementary Figure S2
). The NP fabrication process that allows preparation of very small drug delivery NPs with remarkable control over size and monodispersity and with excellent scalability is unprecedented and offers clear advantages over many other nanomedicine preparation methods.
Aiming to formulate solid silica NCs without aggregation, we tested the lyophilization of silica NCs in the presence of various lyoprotectants (). We found dextrose was overall the best lyoprotectant for silica NC. Silica NCs lyophilized in 1 mL 5% dextrose solution (known as D5W, routinely used for drug administration in clinic) resulted in solid formulation of silica NCs with essentially no change of particle sizes after lyophilization and re-constitution in water ().
Recent studies showed that silica NPs can decompose in blood within a few days,35, 36
suggesting that this class of NPs can be eliminated by either hepatic or renal clearance,37, 38
thereby minimizing concerns for cumulative tissue damage and associated toxicity. In vitro
study (MTT assay; Supplementary Table S4
) showed almost no toxicity of blank silica NPs (IC50
>1 mM). Acute in vivo
toxicity experiments were performed after i.v. administration of 50 nm silica NPs in C57BL/6 mice at very high dose up to 250 mg/kg. There was no mortality or deterioration under general conditions observed in any of the groups. In addition, there were no treatment related clinical signs and change of body weights. Representative sections of various organs taken 24 h after injections from control mice receiving PBS and mice receiving silica NPs were stained by hematoxylin and eosin, and evaluated by an independent pathologist (). The absence of immune or inflammatory reactions after NC administration supports their lack of toxicity. To facilitate faster degradation, we prepared bis-silane agents containing pH-sensitive ester (8
) or orthoester domain (9
). They can be very successfully incorporated to silica NC to make mono-disperse silica NC (entries 23–26, ; supplementary Figure S7
). These silica NC fabrication methods are not only independent of agents, but are also independent of linker (e.g.
, in the context of using 7
, entries 20–21, ) or addition of other silane reagents (e.g.
, in the context of using degradable (8
) or pH-sensitive (9
) silane agent, entries 23–26, ). Study of the in vivo
degradation and clearance of regular silica NCs and 8
-containing silica NCs are underway.
Figure 5 Histopathology of mouse tissues following an intravenous injection of silica nanoparticles via a tail vein. Representative sections of various organs taken from control mice receiving PBS and mice receiving 250 mg/kg 50 nm blank silica nanoparticles 24 (more ...)