Multifunctional Mesoporous Silica Nanoparticles Based on Charge

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Multifunctional mesoporous silica nanoparticles based on charge-reversal plug gate nanovalves and aciddecomposable ZnO quantum dots for intracellular drug delivery Jing Zhang, Dan Wu, Mengfei Li, and Jie Feng ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b08460 • Publication Date (Web): 10 Nov 2015 Downloaded from http://pubs.acs.org on November 19, 2015

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ACS Applied Materials & Interfaces

Multifunctional mesoporous silica nanoparticles based on charge-reversal plug gate nanovalves and acid-decomposable

ZnO

quantum

dots

for

intracellular drug delivery Jing Zhang*, Dan Wu, Meng-Fei Li, and Jie Feng*. College of Materials Science & Engineering, Zhejiang University of Technology, Hangzhou 310014, China KEYWORDS: mesoporous silica nanoparticles, ZnO quantum dots, pH-responsive, chargereversal, drug delivery system (DDS)

ABSTRACT: A novel type of pH-responsive multifunctional mesoporous silica nanoparticles (MSNs) was developed for cancerous cells drug delivery and synergistic therapy of tumor. MSNs were covered with a kind of cell-penetrating peptide, deca-lysine sequence (K10), to enhance escaping from the endosomes. After K10’s primary amines were reacted with citraconic anhydride to form acid-labile β-carboxylic amides, ZnO quantum dots (ZnO QDs) were introduced to cap MSNs via electrostatic interaction. The obtained ZnO@MSN drug delivery system (ZnO@MSN DDS) achieves “zero-premature” drug release under physiological

ACS Paragon Plus Environment

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environment. But once the DDS is transferred to cancerous cells’ acidic endosome, ZnO QDs would rapidly dissolve and the acid-labile amides on the side chain of K10 would hydrolyze to regenerate primary amines, resulting in the uncapping of the MSNs and expose of the cellpenetrating peptide K10. The regenerated K10 could help the DDS escape from endosome and release the loaded drugs inside cells efficiently. At the meantime, on account of the cytotoxicity of ZnO QDs at their destination, the ZnO@MSN DDS may achieve synergistic antitumor effect to improve the therapeutic index.

1. INTRODUCTION Drug delivery methods always play an important role in the therapeutic index of drug.1 Because most traditional anticancer drugs cannot tell cancerous cells from healthy ones, a large amount of drug delivery system (DDSs) were designed to realize selectively release of drug at tumor site based on enhanced permeability and retention effect (EPR effect).2-4 However, most DDSs always have disadvantages that the loaded drug suffered unexpected sustained premature release before reaching their destination. Thus, it is of great importance to develop smart DDSs that can achieve explosive release of drug at the target sites while encapsulating the drugs efficiently during circulation. Recently, surface functionalized mesoporous silica nanoparticles (MSNs) have been emerged as excellent DDSs for their “zero-premature” drug release and sensitive stimulus-responsive controlled drug release properties.5,6 In previous studies, various gatekeepers such as nanoparticles (NPs),7-9 organic molecules,10-12 and supramolecular assemblies,13-15 have been used to cap the pores of MSNs that can respond to different stimuli, for instance, pH, redox,


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enzymatic activity, irradiation, and electrostatic interaction. Among these triggers, changing pH is highly attractive due to the more acidic environment around tumors and inflammatory tissues comparing with normal tissues as well as the even lower pH values in endosome and lysosome.16,17 This motivated us to fabricate delivery vehicles which allow the adjusted drug release that could respond to physiopathological pH signals. A few pH-responsive DDSs based on MSN have been reported by previous studies. However, most of systems need harsh pH environment to drive the drug release (pH 3~4 or pH~10), 18-21 limiting their application in biological field. To address this challenge, zinc oxide quantum dots (ZnO QDs) attracted a lot of research interest due to its adequate acid-responsibility. ZnO QDs is stable under conditions of pH 7.4, but can immediately dissolve when pH is less than 5.5. Moreover, besides the unique optical and electronic properties of quantum dots, ZnO QDs show distinct advantages in terms of simple preparation method, inexpensive property and exhibiting cytotoxicity when accumulate inside cells involving dissolution into Zn2+.22 These superiorities make the ZnO QDs a satisfactory candidate as gatekeeper to fabricate pH-responsive MSN DDS. Furthermore, ZnO QDs have been proved that they exhibit more cytotoxicity on cancer cells than normal ones.23 Thus, we would think use of ZnO QDs as the nanovavles in order to achieve the synergistic therapy of tumor. As the digestive system of cells, lysosome contains large amounts of acid hydrolases, which will lead degradation of inclusions (drug and carrier).24 In order to avoid enzymatic degradation, the DDS must escape from endosome and enter cytoplasm before the fusion of endosome and lysosome, which is called endosome escape (endosomal escape).25 It is well documented that having cell-penetrating peptide on the surface of NPs can promote the NPs to cross cell membrane.26 Here, we expect the cell-penetrating peptide K10 (deca-lysine sequence) to help the


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DDS to escape from endosome. As illustrated in Scheme 1, MSNs were firstly covered with K10. After K10’s primary amines were amidized to form acid-labile β-carboxylic amides, ZnO QDs were introduced to lid the pores of the MSNs via electrostatic interaction. Once the DDS is transferred to cancerous cells’ acidic endosome,27 ZnO QDs would rapidly dissolve and the acidlabile amides on the side chain of K10 would hydrolyze to regenerate primary amines, resulting in uncapping of the MSNs and expose of the cell-penetrating peptide K10. The regenerated K10 could help the DDS rapidly escape from endosome and traverse into the cytoplasm, avoiding from degradation of loaded drug by acid hydrolases in lysosomes and realizing efficient drug delivery. At the meantime, on account of the cytotoxicity of Zn2+,22 intracellular dissolution ZnO QDs into Zn2+ is helping achieve synergistic antitumor effect to improve the therapeutic index. Scheme 1. Schematic Illustration of Intracellular Drugs Release from ZnO@MSNa


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2. Experimental Section 2.1. Preparation of Azide Modified K10 (K10-azide). The peptide K10-azide was synthesized according to standard Fmoc-typed solid phase peptide synthesis. 2-Chlorotriyl chloride resin (loading amount: 1.29 mmol g-1) was chosen as the starting resin and 4 equivalents of FmocLys(Boc)-OH were coupled to the resin in DMF with 6 equivalents of diisopropylethylamine (DIEA). Then the Fmoc moieties were removed by rinsing the resin with 20% piperidine/DMF (v/v) and the subsequent coupling between the resulting amino groups and carboxylic groups of the next amino acid was achieved by adding 4 equivalents of Fmoc-Lys(Boc)-OH, 4 equivalents


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of

o-benzotriazol-N,N,N´,N´-tetramethyluronium

hexafluorophosphate

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(HBTU),

and

6

equivalents of DIEA in DMF solution. Repeating the above procedure of amino acid conjugation until the last lysine was connected. Azido-glycine was used to terminate the peptide sequence. After the completion of peptide synthesis, a cleavage agent (v: TFA/v: triisopropylsilane/v: H2O = 95/2.5/2.5) was utilized to cut the peptide sequence from the resin while remove all the side protective groups. The crude peptide was isolated from the cleavage agent suspension by filtration, concentration and precipitation (in cold ether). The collected white powder was dissolved in de-ionized (DI) water and then freeze-dried in vacuum. 2.2. Synthesis of MCM-41 Type MSNs, Amino-Functionalized MSNs (MSN-NH2) and Alkyne Modified MSNs (MSN-alkyne). The synthesis procedure was according to our previous study.6,28 The detail of the method was offered in supporting information. 2.3. Synthesis of MSN-K10. The coupling of MSN-alkyne (300 mg) and K10-azide (300 mg, 0.217 mmol) were catalyzed by CuSO4·5H2O (150 mg, 0.6 mmol) and sodium ascorbate (238 mg, 1.2 mmol) in DI water under N2 atmosphere at 25oC for 3 days. The resulting solid was centrifuged 8000 r/min for 5 min. MSN-K10 was obtained after washed thoroughly with water and methanol for 3 times respectively, and finally dried under vacuum. 2.4. Synthesis of MSN-K10(cit). The functionalization of citraconic anhydride was according to our previous study.28 The detail of the method was offered in supporting information. 2.5. Synthesis of ZnO QDs. 0.408 g (1.86 mmol) Zinc acetate was dissolved in 10 mL anhydrous ethanol and refluxing at 68 oC for 1.5 h. Then the solution was cooled to room temperature and 0.231 g (4.11 mmol) KOH dissolved in 5 mL anhydrous ethanol was added dropwise. After reacting for 1 h, 1 mL APTES in 5 mL anhydrous ethanol and 0.5 mL DI water


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were added and the solution was stirred at 25oC for 2 h. ZnO QDs were centrifuged (11000 r/min, 5 min), washed with anhydrous ethanol and dispersed in DI water. 2.6. Synthesis of Dox Loaded ZnO@MSN. 100 mg MSN-alkyne and 10 mg Dox were dispersed in 10 mL phosphate buffered saline (PBS) (10 mM, pH 7.4). After stirring at room temperature overnight, K10-azide (100 mg, 0.07mmol), CuSO4·5H2O (50 mg, 0.2 mmol) and sodium ascorbate (79.3 mg, 0.4 mmol) were added and the mixture was stirred under N2 atmosphere at 25oC for 3 days. The NPs were centrifuged and then suspended in 15 mL anhydrous DMF solution and reacted with 1.5 mL citraconic anhydride overnight. After centrifugation at 8000 r/min for 5 min, carefully washed with DI water for one time, the particles were suspended in 5 mL DI water and added to ZnO QDs aqueous solution dropwise. The mixture was reacted at 25 oC for 5 h. The resulting NPs was collected though centrifugation at 8000 r/min for 5 min. After washing thoroughly with DI water, Dox loaded ZnO@MSN were dried under vacuum. The amounts of ZnO QDs in the ZnO@MSN DDS was measured by Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES). Firstly, ZnO@MSN and ZnO were accurately weighted. After dissolved in 40% HF aqueous solution, the samples were heated to evaporate the silicon tetrafluoride and the excess HF aqueous solution, respectively. Then 1% HNO3 aqueous solution was added to dissolve the resulting solid residue in a certain concentration. The concentration of Zn2+ in the solution was calculated based on the spectral line intensity at 202.548 nm, calibrated by a standard curve of Zn2+.


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The amounts of ZnO QDs in the ZnO@MSN can be calculated using the following equation: (m@ and m represents the weight of ZnO@MSN and ZnO QDs, m(@) and m() represents the weight of Zn2+ in ZnO@MSN and ZnO QDs.) m ∗ m(@) m@ ∗ m() 2.7. In Vitro Release Experiments. The in vitro release behavior of Dox from MSN and the obtained Dox loaded ZnO@MSN was investigated. At 37 oC 3 mg of the NPs were suspended in three different pH (pH 7.4 phosphate buffer, pH 5.0 acetate buffer and pH 2.0 glycine buffer), respectively. The fluorescence of the medium was measured by spectrofluorophotometer (RF530/PC, Shimadzu) at given incubation time intervals. (λex=470 nm, slit widths: 10 nm) 2.8. Cells culture. Dox loaded ZnO@MSN was incubated with HepG2 cells (liver hepatocellular carcinoma) over time. The detail co-incubation procedure were carried out according to our previous study29 except that HepG2 cells were incubated in a 96-well plate (5000 cells per well) and Dox loaded ZnO@MSN was dispersed in culture medium at concentration of 50 µg/mL. The cells were observed under fluorescent inverted microscope (Nikon Ti). 2.9. MTT Assay. The cell viabilities of HepG2 cells incubated with different NPs were studied to investigate the therapeutic efficiency of the ZnO@MSN DDS. The detail procedure was carried out according to our previous study29 except that HepG2 cells were incubated in a 96-well plate (5000 cells per well). 3. RESULTS AND DISCUSSION


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In this study, MSNs was synthesized according to the literature6 and the mesoporous structure was confirmed by transmission electron microscope (TEM) (Figure 1A) and BarrettJoyner-Halenda (BJH) analyses (Table 1). From Figure 1A, highly ordered lattice array of the obtained MSNs could be clearly visualized. Dynamic Light Scattering (DLS) measurement (Figure 1B) shows that the average diameter of MSNs is ~200 nm. As represented in Scheme 2, the MSNs was then functionalized with ATPES28,30 to produce MSN-NH2. And after subsequent reaction with propargyl bromide, MSN-alkyne was obtained. The surface functionalization of MSNs was characterized by Fourier Transform-Infrared Spectroscopy (FT-IR) (Figure 2). The typical absorption peak at 2120 cm-1 which is attributed to alkyne groups verifies the successful synthesis of MSN-alkyne (Figure 2C).

Figure 1. (A) TEM micrograph of MSNs; (B) Size distribution of MSNs; (C, D) Low- and high magnification TEM micrographs of ZnO QDs. Table 1. BET and BJH Parameters of Different Nanoparticles.


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Scheme 2. The Schematic Illustration of Synthetic Route


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Figure 2. FT-IR spectra of MSNs (A), MSN-NH2 (B), MSN-alkyne (C), MSN-K10 (D), and MSN-K10(cit) (E). Thereafter, we functionalized the surface of MSN-alkyne with K10. K10-azide was synthesized according to standard solid phase methodology based on Fmoc chemistry. ESI-MS spectrum of K10-azide proved the correct structure of the peptide (Figure 3). K10-azide was anchored onto the surface of MSNs via Cu-catalyzed azide–alkyne cycloaddition (CuAAC) click chemistry reaction. As displayed in Figure 2D, the strong absorption peak at 1680 cm-1, which is attributed to absorption band of amide moieties in K10, and disappearance of the typical absorption peak of alkyne groups at 2120 cm-1 mean that K10 has been successfully anchored onto MSNs. To achieve surface charge switchable ability of the nanovector under endosomal environment, MSN-K10 was reacted with citraconic anhydride to form acid-labile amides, resulting in negatively surface charged MSN-K10(cit). From Figure 2E, we can find the appearance of absorption band at 1725 cm-1, belonging to C=O in carboxyl groups. At the same


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time, it can be seen that the surface charge of the NPs was changed from +26 mV to -24 mV (Figure 4), indicating the successful modification of citraconic anhydride.

Figure 3. ESI-MS spectrum of K10-azide.

Figure 4. Zeta potential measurement of different particles. Subsequently, aminopropyl-functionalized and water-dispersible ZnO QDs were synthesized. TEM images show the average size of ZnO QDs was ~3nm (Figure 1C, D). Based on electrostatic interaction, negatively charged MSN-K10(cit) (ζ-potential = -24 mV) were capped


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by positively charged ZnO QDs (ζ-potential = +25 mV). As illustrated in Scheme 1, under endosome acidic environment, ZnO QDs would rapidly dissolve, and the negatively charged MSN-K10(cit) would reverse to positively charged MSN-K10 via hydrolysis of the acid-labile amides to expose cationic K10 on the outer face of the MSNs. The interaction between MSNs and ZnO QDs was confirmed by TEM microscopy. Figure 5A and 5B show the TEM images of MSNs after capping with ZnO QDs. Compared with Figure 1A, dark spots on the exterior edges of the ZnO@MSN shown in the TEM micrographs, indicate the aggregation of ZnO QDs on the surface of MSNs. These results were consistent with the ones demonstrated in Figure 6. As can be seen in Figure 6A-D, yellow color can be clearly visualized under UV light irradiation in the condition of ZnO QDs and ZnO@MSN while no color can be seen in MSNs before capping. The presence of ZnO QDs was also confirmed by spectrofluorophotometer (Figure 6E, F). In the case of the uncapped MSNs, no fluorescence was detected between 450 nm and 700 nm. In contrast, the fluorescence spectrum of ZnO@MSN shows an obvious absorption peak at about 525 nm, which is attributed to the fluorescence of ZnO QDs. Furthermore, we can find that the surface charge of the NPs has turned to +25 mV after capping with ZnO QDs (Figure 4), also indicating the successful fabrication of ZnO@MSN DDS. The amounts of ZnO QDs was 31.03 wt % determined by ICP-OES.


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Figure 5. (A, B) Low and high magnification TEM micrographs of ZnO@MSN; (C, D) TEM and SEM micrographs of ZnO@MSN after incubation in pH 5.0 buffer solution.

Figure 6. Digital photographs of ZnO QDs (A), ZnO QDs under UV light irradiation at 365 nm (B), ZnO@MSN (left) and MSNs (right) (C), ZnO@MSN (left) and MSNs (right) under UV light irradiation at 365nm (D); (E) fluorescence spectra of ZnO QDs incubated at different pHs; (F) fluorescence spectra of MSNs and ZnO@MSN incubated at different pHs. Since the pH sensitivity of the obtained ZnO@MSN DDS is based on two mechanism, dissolution of ZnO QDs and hydrolysis of the acid-labile amide bond connecting citraconic anhydride to K10, we investigated the dissolution of ZnO QDs and charge-conversion ability of


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MSN-K10(cit) under mildly acidic condition, respectively. The dissolution of ZnO QDs was investigated by measuring the fluorescence intensity of the quantum dots and ZnO@MSN immediately after incubation at different pHs. As illustrated in Figure 6E and F, fluorescence absorption peak of ZnO QDs could be detected at pH 7.4 but it was disappeared at pH 5.0, confirming that ZnO QDs could be stable at pH 7.4 while immediately dissolve when pH is changed to 5.0.

Figure 7. Zeta potentials of MSN-K10(cit) after incubated in different pH solutions at 37 oC for different time intervals. At the same time, the charge conversion behavior of MSN-K10(cit) was investigated. We incubated the NPs in pH 7.4 phosphate buffer and pH 5.0 acetate buffer, respectively, to imitate the physiological environment and endosomal environment. As shown in Figure 7, initially, MSN-K10(cit) was negatively charged both at pH 5.0 and 7.4. With the increase of incubation time, the zeta potential of the NPs incubated at pH 5.0 increased significantly to positive within 20 min and reached +19.2 mV after 2 h, verifying the transforming of carboxyl groups to amino groups. In contrast, although the zeta potential of the NPs showed slowly increasing from -30.2 mV to -24.1 mV when incubated at pH 7.4, it maintained negatively within 2 h charged.


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Considering that endosomal environment is mild acidic, the change conversion of the ZnO@MSN DDs could be achieved in endosome and thus enhance the endosomal escape.

Figure 8. Release profiles of ZnO@MSN DDS (A) and MSN (B) after incubated in different pH solutions at 37 oC for different time intervals. To demostrated the pH-responsive uncapping efficiency of the ZnO@MSN DDS, in vitro release from ZnO@MSN DDS at different pH values was monitored. Dox was loaded before the NPs were covered with K10 for that the chains of peptide may hinder drug molecules from getting into the pores of MSNs. The drug loaded in the ZnO@MSN DDS was ~1 wt % as determined by spectrofluorophotometer. According to the drug release studies shown in Figure 8A, at pH 7.4, Dox released from ZnO@MSN DDS could be negligible after 48 h, indicating the efficient packing of drug molecules in MSNs capped with ZnO QDs. On the contrary, as


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incubation at pH 5.0, Dox was released fast from the DDS, confirming that the ZnO@MSN DDS could response to the pH signal under endosomal environment and realize intracellular drugs release. However, compared to the immediate dissolution of ZnO QDs under mild acidic environment which was proved above, the release behavior did not show explosive release but sustained release due to the hindrance of K10 on the surface of MSNs. This is desirable because more drugs could be protected before endosome escape and thus be released in cytoplasm. Release profiles revealed that about 34 % of Dox was released at pH 5.0 within 12 h and then changed slowly over time for the electrostatic interaction between the positive Dox molecular and the negative silanol moieties in the pore reservoirs.31,32 Therefore, we incubated the NPs in pH 2.0 glycine buffer in order to eliminated the undesirable interaction between Dox and the silica. And we can see that about 86 % of Dox could be released within 48 h. Furthermore, the ZnO@MSN was subjected to TEM and SEM microscopy after incubation at pH 5.0 for 48 h. The smooth surface of the MSNs indicates the perfect detaching of the ZnO QDs (Figure 5C, D). For comparison, release behavior of Dox from MSN was investigated. From Figure 8B, we can see that release behavior was pH dependent, but did not show zero-release at pH 7.4 since there is no gatekeeper on the surface. Moreover, because Dox loaded MSN cannot be washed thoroughly after drug loading, otherwise the drug loaded in the pores of MSN would be wash out during the washing step, there existed a large amount of Dox absorbed on the surface of MSN. Thus the drug was released very fast at the beginning and total amount of drug loaded and released from MSN would be higher than that of ZnO@MSN.


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Figure 9. Fluorescent inverted microscope micrograohs of HepG 2 cells after incubation with Dox loaded ZnO@MSN for 2 h (A, B), 4 h (C, D) and 8 h (E, F): (left) bright field micrograohs; (right) red fluorescence micrograohs. (Scale bar: 20 µm). Cellular uptake and intracellular drug release behavior were studied by fluorescent inverted microscope. After incubation with HepG 2 cells over hours, ZnO@MSN DDS was gradually internalized into the cells. The clearly observable red fluorescence which is attributed to Dox indicated the released Dox was localized mainly in the nucleus (Figure 9), implying efficient delivery of the loaded drugs. Under the endosomal environment, ZnO QDs dissolved immediately and the amides on the side chain of K10 hydrolyzed to regenerate the amines and thus enhanced endosome escape of the DDS, avoiding degradation of loaded drugs by acid hydrolases in lysosomes.


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Figure 10. (A) Viabilities of HepG 2 cells after being incubated with Zno QDs and Zn2+ for 48 h, respectively; (B) Viabilities of HepG 2 cells after incubation with MSN-K10(cit), ZnO@MSN, Dox loaded ZnO@MSN and free Dox for 48 h, respectively. The viabilities of HepG 2 cells incubated with of ZnO QDs and the corresponding concentrations Zn2+ ions (as ZnCl2) were investigated. As shown in Figure 10A, both samples showed similar trend and decreased the cell viability below 50 % when their concentration surpassed 25 µg/mL. This suggests that ZnO QDs exhibit cytotoxicity when accumulate inside cells involving dissolution into Zn2+. Then MTT assay was carried out to investigated cell viabilities of HepG 2 cells incubated with MSN-K10(cit), ZnO@MSN, Dox loaded ZnO@MSN and free Dox. As shown in Figure 10B, MSN-K10(cit) had no obvious cytotoxicity when its concentration reached 100 µg/mL, whereas the cell viability decreased to 42 % in the presence of


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ZnO@MSN at the same concentration, because ZnO QDs were dissolved into ionic Zn2+ intracellularly. As reported by literature, Zn2+ ions would induct the production of reactive oxygen species and involves in peroxidation of lipid and damage of DNA33-36, resulting in its cytotoxicity. Figure 10B also demonstrate that the cell viability with Dox loaded ZnO@MSN decreased below 50% at concentration of 50 µg/mL, which is more effective than free Dox. We think that this is owed to both the loaded Dox and the dissolution of ZnO QDs, exhibiting synergistic antitumor effect. 4. CONCLUSION In conclusion, we demonstrated a dual pH-sensitive DDS based on intracellular surface charge reversal of MSNs and dissolution of ZnO QDs. The drug loaded in the MSNs can be released under endosome acidic environment. Using of ZnO QDs as gatekeeper of MSNs achieves synergistic antitumor effect. The cell-penetrating peptide K10 at the surface of MSNs could help the DDS escape from endosomes and deliver drugs efficiently. The multifunctional ZnO@MSN DDS improved the therapy efficiency. ASSOCIATED CONTENT Supporting Information Materials, synthesis of MCM-41 Type MSNs, synthesis of MSN-NH2, synthesis of MSN-alkyne, synthesis of MSN-K10(cit), FT-IR and zeta potential characterization. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author


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* Email: [email protected] (J.Z.) and [email protected] (J.F.). Funding Sources National Natural Science Foundation of China (21404091 and 51172206) and Zhejiang Provincial Education Department research (Y201432594). Notes The authors declare no competing financial interest. ACKNOWLEDGMENT We acknowledge the financial support from National Natural Science Foundation of China (21404091 and 51172206) and Zhejiang Provincial Education Department research (Y201432594). EXPLANATORY NOTES (a) Under endosomal environment, ZnO QDs rapidly dissolved and the acid-labile amides on the side chain of K10 hydrolyze to regenerate primary amines, resulting in uncapping of the MSNs and expose of the cell-penetrating peptide K10. The regenerated K10 could help the DDS rapidly escape from the endosomes and traverse into the cytoplasm, avoiding from degradation of loaded drug by acid hydrolases in lysosomes and realizing efficient drug delivery. REFERENCES (1) Bakken, E. E.; Heruth, K. Temporal Control of Drugs: An Engineering Perspective Ann. N. Y. Acad. Sci. 1991, 618, 422-427.


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Multifunctional Mesoporous Silica Nanoparticles Based on Charge

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