OPEN Multifunctional Enveloped Mesoporous

SUBJECT AREAS:
Silica Nanoparticles for Subcellular
DRUG REGULATION
CANCER THERAPY
Co-delivery of Drug and Therapeutic
Received
Peptide
30 April 2014
Guo-Feng Luo, Wei-Hai Chen, Yun Liu, Qi Lei, Ren-Xi Zhuo & Xian-Zheng Zhang
Accepted
25 July 2014
Key Laboratory of Biomedical Polymers of Ministry of Education & Department of Chemistry, Wuhan University, Wuhan 430072,
Published P. R. China.
14 August 2014

A multifunctional enveloped nanodevice based on mesoporous silica nanoparticle (MSN) was delicately
designed for subcellular co-delivery of drug and therapeutic peptide to tumor cells. Mesoporous silica
Correspondence and MCM-41 nanoparticles were used as the core for loading antineoplastic drug topotecan (TPT). The surface
requests for materials of nanoparticles was decorated with mitochondria-targeted therapeutic agent (Tpep) containing
should be addressed to triphenylphosphonium (TPP) and antibiotic peptide (KLAKLAK)2 via disulfide linkage, followed by
X.-Z.Z. (xz-zhang@ coating with a charge reversal polyanion poly(ethylene glycol)-blocked-2,3-dimethylmaleic
whu.edu.cn)
anhydride-modified poly(L-lysine) (PEG-PLL(DMA)) via electrostatic interaction. It was found that the
outer shielding layer could be removed at acidic tumor microenvironment due to the degradation of DMA
blocks and the cellular uptake was significantly enhanced by the formation of cationic nanoparticles. After
endocytosis, due to the cleavage of disulfide bonds in the presence of intracellular glutathione (GSH),
pharmacological agents (Tpep and TPT) could be released from the nanoparticles and subsequently induce
specific damage of tumor cell mitochondria and nucleus respectively with remarkable synergistic antitumor
effect.

D
uring the last decades, intense efforts have been made to construct various drug nanocarriers based on
metals1, metal oxides2–4, micelles5, and liposomes6 for tumor-targeted drug delivery. However, there are
many physiological barriers for the nanocarrier reaching the particular target site, including circulating
from the blood compartments to the tumor extracellular matrix, sticking to tumor-cell membrane for fast cell
internalization, releasing the encapsulated cargo within cells, and targeting to subcellular sites of action in turn.
Recently, enveloped nanodevice of programmatically packing the nano-core with different functional groups has
been proposed to surmount all these physiological barriers7,8. For example, PEGylation is widely used as a stealthy
layer for avoiding quick recognition of the carriers by the immune system and thereby extending blood circulation
time9,10. Moreover, for resisting protein adsorption, negatively charged carriers can be used to block the inter-
action with cell membrane due to electrostatic repulsion11. Nevertheless, positively charged nanoparticles can
enter cells easily because of their high affinity to negatively charged cell membrane, although they display rapid
clearance from blood circulation12. Thus, it is highly desirable to fabricate PEGlated and charge switchable
nanoparticles that exhibit good shielding effect against normal cells with a prolonged circulation time, but alter
their surface charge at tumor site and become sticky targeting to tumor cells. Positive nanoparticles were reported
with the capability to strip stealth layer at acidic environment and re-expose the positively charged surface for
improving tumor cell uptake by employing a tumor acidity-sensitive PEGlated anionic polymer13.
For internalization of nanoparticles by tumor cells, it is essential to liberate therapeutic agents into cytosol or
even specific subcellular organelle where most therapeutic agents accumulate and take effect. In contrast to single-
agent chemotherapy, combining two or more drugs with synergistic therapeutic effect has been employed for
more efficient tumor treatment. As the cellular heart where hereditary material and the transcription machinery
reside, the cell nucleus has been demonstrated to be the final targeting destination of various therapeutic agents
(e. g. genes and antitumor drugs)14,15. Thence, traditional combination therapies mainly focused on co-delivery of
drug/drug or gene/drug to the cell nucleus16–21. Besides, mitochondria, the powerhouse of eukaryotic cells, also
play crucial roles in diverse essential cellular functions for cell viability and proliferation as well as programmed

SCIENTIFIC REPORTS | 4 : 6064 | DOI: 10.1038/srep06064 1

this ity32 allows MSNs to react with diverse agents.3-dimethylmaleic anhydride Figure 1 | Schematic design of the delivery process: (I) multifunctional enveloped nanocarrier under physiological condition. for by cleavage of the disulfide bonds. including macrocyclic promotes its internalization by tumor cells with superior efficacy in compounds33–36.26. was reported to spe. (II) charge-conversional detachment of PEGlated corona in acidic tumor microenvironment. SCIENTIFIC REPORTS | 4 : 6064 | DOI: 10.8)50.29) and linkages between amines and DMA51) accompanied by removing the fluorescent dyes30. (III) adsorption-interaction between positively charged nanoparticles and cell membrane. In addition. Thereafter. to nucleus and mitochondria respectively for synergistic antitumor inducing the dysfunction of mitochondria and killing tumor cells.nature. an antineoplastic drug topotecan (TPT). (V) specific binding and disrupting of mitochondria. se the viscous cytosol to the mitochondria sites. carboxyanhydriade (Z-Lys-NCA) initiated by the terminal amino Cationic antibiotic peptide.com/scientificreports cell death22. www. due to nanoparticles (MSNs) are competitive carriers for loading drugs in the intrinsic tumor extracellular acidity (pH . was loaded in the mesopores was synthesized by Fmoc-based solid-phase synthesis manually52 and of MCM-41 type MSN. charge of the enveloped MSN changes to positive. TPT loaded in the inner core can be released in cells. followed by deprotection of the carboben- cially disrupt the mitochondrial membrane and initiate apop.41. normal cells because of the coexistence of PEG polymers and Among different drug delivery systems (DDSs). For mitochondria-targeted delivery. PEG-PLL(DMA) on the surface of nanoparticles results in ledge. Meanwhile. And the multifunctional enveloped MSN TPP was conjugated to the last N-terminal group of the peptide. (IV) intracellular glutathione-triggered TPep & TPT release. 6. The modular therapeutic peptide sequence K-(KLAKLAK)2-C enzyme topoisomerase I inhibitor47. In the presence of intracellular nanoparticles42–45. a new glutathione (GSH). the lipophilic triphenyl. as a nuclear MSN. Conjugating these two domains would give rise to a to irreversible lethality on cells. Once arriving at solid tumor site. In this report.31 have been encapsulated in MSNs for controlled protecting layer through electrostatic repulsion. in which the surface drug release. Accordingly. the vast surface functionalization capabil. biomacromolecules40. the pH the pores due to their tunable pore sizes and large pore volumes23. layer. (KLAKLAK)2. and even therapeutic drugs46. delivering therapeutic agents. leading to much enhanced therapeutic effect together with TPep. group of PEG (Mw 2000). mesoporous silica negatively charged chains. dendrimers38. and CPT28. for the first time.39. The outer protecting layer PEG-PLL(DMA) was obtained by the phosphonium (TPP) cation as mitochondrial anchor has been ring-opening polymerization of e-benzyloxycarbonyl-L-lysine N- demonstrated to effectively deliver payloads into mitochondria48. The specific damage of mitochondria.1038/srep06064 2 . TPep could be released from the nanoparticles multifunctional enveloped MSN was designed. TPT27. polymers37. The was obtained by programmed packing of mitochondria-targeted molecular weight was analyzed by the MALDI-TOF-MS and the therapeutic agent and PEGlated charge conversional shielding purity was analyzed by HPLC (Table S1). zyloxy groups and modification with 2. Results Design of the multifunctional enveloped MSN. Incorporation of leus respectively for combination therapy. A triggered charge conversion occurs (due to the hydrolysis of amide variety of pharmaceutical drugs (DOX25. which could traver- two different therapeutic agents targeting to mitochondria and nuc.24. there is no report on co-delivering different therapeutic agents prolonged blood circulation time and good shielding effect against to different subcellular organelles based on a single nanocarrier. To the best of our know. renders the possibility of combining mitochondria-targeting therapeutic agent TPep. which would lead totic death49. therapy. the lipophilic TPP intracellular co-delivery of antitumor drug and therapeutic peptide would targetedly transport (KLAKLAK)2 to the mitochondria. As illustrated in Preparation and characterization of the multifunctional enveloped Figure 1.

finally reached to about 24 mV after 5 h. DMA blocks. as shown in Figure 2G. Gel permeation conversion property in acidic condition due to the degradation of chromatographic (GPC) measurement of PEG-PLL exhibited a sin. MSN-TPep. an increased weight loss was obtained: from negative to positive. Finally.7 mV. TPep(Rh B) and MSN-TPep(Rh B)/PEG-PLL(DMA) were co- Subsequently. the zeta potential of MSN- gle sharp peak at Mw of 6. MSN-TPep/PEG-PLL(DMA). which was nanoparticle was expected to exhibit a negative-to-positive charge confirmed by 1H NMR (Supplementary Figure S3A).8. and onance (1H NMR) of PEG-PLL(Z) was revealed in Figure S2. the hydrolysis of the amide after each functionalization step. there was weak red fluorescence in cells at pH 7. The ant-directed self-assembly procedure. incubated with KB cells (human mouth epidermal carcinoma cells) duced by disulfide exchange reaction. (C) DLS analysis of MSN. surements. As shown in Figure 2G. (B) TEM image of MSN.8. (D) Zeta-potentials of (a) MSN. the zeta potential remained at around 212 mV within 5 h.2 mV). mainly desorption measurements (Figure 2E) revealed that the BET surface attributed to the shielding effect of the unrevoked PEGlated layer. diameter was calculated as .7% for MSN-SH. the cell-interactive cationic nanoparticles were obtained. As shown in interaction for preparing negatively charged MSN-TPep/PEG. (G) Zeta- potential changes of MSN-TPep/PEG-PLL(DMA) as a function of incubation time at pH 7. TPep containing a free cysteine residue was intro. which 33. Scanning electron microcopy hydrolysis of amides linkages between amines and DMA made the (SEM) (Figure 2A) and transmission electron microscopy (TEM) outer corona detachable under tumor acidic environment. that free TPep(Rh B) was unable to penetrate into cells. ity-responsive anionic PEG-PLL(DMA) was anchored to the surface there was a appreciable difference in cellular uptake of MSN- of positively charged MSN-TPep nanoparticle by electrostatic TPep(Rh B)/PEG-PLL(DMA) at different pHs. www.4. (A) SEM image of MSN. at pH 7. MSN-SH was obtained with a slightly increased reticulum structures of mitochondria were observed as green but still negatively charged surface (223.4 and 6. 27. As shown in Figure 3A and Figure 3B. nearly charged nanoparticle (35.3 nm (Figure 2B). (d) MSN-TPep/PEG-PLL(DMA). As summarized in Figure 2D. MCM-41 type of MSN was prepared by a base-catalyzed surfact. confocal laser scanning (Supplementary Figure S4) were monitored by zeta potential mea. Dynamic light scat- tering (DLS) data (Figure 2C) presented a relatively larger diameter Evaluation of the tumor-acidity-triggered targeting of the due to the hydrated layer surrounding the particles with a narrow multifunctional enveloped MSN. area. However.90 3 103 g/mol (PDI: 1. The molecular firming the successful modification.3). the tumor acid.7 mV) to positive (5. 13.29-dipyridyl disulfide. leading to electrostatically removing of the Figure 2 | Characterization of the multifunctional enveloped MSN. by thermogravimetric bonds at pH 6. MSN-SH. The mercapto groups. The 1H NMR TPep/PEG-PLL(DMA) increased obviously from negative spectrum of PEG-PLL(DMA) (Figure S3B) confirmed that .8 and the amino groups in PLL block were converted to the amide bonds.2% for MSN-TPep/PEG-PLL(DMA) respectively.4 mV) within 90 min at pH 6. SCIENTIFIC REPORTS | 4 : 6064 | DOI: 10. 20.81% of (213. After surface functionalization by TPep(Rh B)) for observing their intracellular distribution. (b) MSN-SH. indicating the inefficient uptake of particles by cells. (F) TGA curves of different nanoparticles. PLL(DMA) nanoparticle (212. In contrast. the zeta potential of ori. BJH pore volume and pore diameter decreased progressively Comparatively. Inset: Nitrogen adsorption2desorption isotherms. Moreover. no red fluorescence was observed at both pH 7. The 1H nuclear magnetic res. Figure 3C.5% for MSN-TPep. To evaluate the tumor particle size distribution. with a monodisperse distribution of the nanoparticles.1038/srep06064 3 . further con- proved the successful synthesis of PEG-PLL(Z).8 resulted in the charge reversal of PEG-PLL(DMA) analysis (TGA) (Figure 2F). gaving rise to positively at different pHs for 4 h. Nitrogen adsorption. (c) MSN-TPep. Free groups were then activated by treating with 2. microscopy (CLSM) was utilized to observe the cellular uptake. (E) Pore size distribution of MSN. weight of PEG-PLL was calculated as 8.nature. And the pore which will be fascinating for tumor targeted delivery.com/scientificreports DMA (Supplementary Figure S1). the TPep agents were tagged with Rh B (defined as ginal MSN was 230.3% for MSN.4 mV). Noted here.14 3 103 g/mol (Mn) and The multifunctional enveloped MSN-TPep/PEG-PLL(DMA) the degree of polymerization was determined to be 48. indicating ing polyanion layer on the external surface. And sub- (Figure 2B) images showed an average diameter of about 120 nm sequently. The consecutive modification processes extracellular acidity triggered targeting. The mercapto fluorescence after stained with Mito Tracker Green FM.4 and 6.4.2 mV) which was very important for coat.

C2 and D2) red fluorescence images. This diffe- fluorescence in cells at pH 6. the fluorescence from red Rh B and green platform for mitochondria-targeted drug delivery. which mitochondria efficiently by its electrophoretic force and lipophili- represents the relative fluorescence intensity corresponding to the city53. (D) for 4 h. (A3. B3. The white arrows indicate the nanoparticles (N).8. rence was ascribed to the specific mitochondria targeting ability of Moreover. the positively charged MSN-TPep(Rh B) nanoparticles were areas were observed in the merged image of cells treated with non- favorable for subsequent cellular uptake. C3 and D3) confocal fluorescence images. And thus the red TPP-conjugated particles (Supplementary Figure S5).4 (A). (A1. which was consistent with the literature report that TPP fluorescence. However. no significant overlapping layer. (A2. (F) TEM images of KB cells treated with MSN-TPep/PEG-PLL(DMA) at pH 7. (C) and 6.4 and (G) at pH 6. www. (D) at pH 7. Figure 3 | CLSM images of KB cells treated with free TPep(Rh B) (A). The ability of mitochondrial targeting provided a good white line in Figure 3D3. (A4. B4.54.com/scientificreports PEGlated protecting layer. B2. SCIENTIFIC REPORTS | 4 : 6064 | DOI: 10. (E) Fluorescence signals corresponding to the white line in D3. C4 and D4) overlay of confocal fluorescence and bright field images.nature. C1 and D1) green fluorescence images.8 (B). (B) and MSN-TPep(Rh B)/PEG-PLL(DMA) (C). As further revealed in Figure 3E. B1. the merged picture of Figure 3D3 showed strong yellow TPP.8 dramatically enhanced (Figure 3D2). indicating the co-localization of red TPep(Rh B) and facilitates the entrance of small molecules or nanosystems into green mitochondria.1038/srep06064 4 . After removal of the PEGlated protecting Mito Tracker matched well.

chondria. C. activity under treatment of free drugs. 7. the tumor acidic microenvironment (pH 6. suggesting that the pores became opened after triggered release behavior55. the mitochondria maintained KB cells incubated with free TPT is almost the same at pH 6. This change can be indicated by JC-1 erated TPep release from the nanoparticles triggered by intracel- probe for distinguishing damaged mitochondria from normal ones.65% Evaluation of the specific mitochondria damage by JC-1 assay and TPT was loaded into the mesopores of the nanoparticles. lular GSH should be responsible for the increased cytotoxicity of As shown in Figure 4. As we know. flow cytometry analysis was carried out to quantita. As shown in upon the trigger of intracellular GSH.8-fold increase of the mean fluorescence acidity-triggered cellular uptake for TPT loaded nanoparticles intensity (MFI) in cells incubated at pH 6. suggesting that these nanoparticles had inappreciable toxic effect To investigate the mitochondrial damage/disruption by released on cells. PLL(DMA) was . A similar phenom. maximum release amount (65% TPep and TPep* up to a concentration of 25 mg/L at different of TPep(Rh B)) was detected. Bio-TEM observation. D. from energy-dispersive X-ray (EDX) (Figure 5F) was detected. co-delivery capability of multifunctional MSNs. CLSM observations revealed a similar tumor- particles.4.8 after incubation for 48 h). www. Evidently. The cell viab- response. both TPT@MSN-TPep*/PEG-PLL(DMA) and respectively). the outer protecting dria) with the prolonged incubation time. a significant silicon signal observed in cells at pH 6. after treating KB cells remained above 90% when they were treated with free with 10 mM GSH at pH 6. Very little particles existed in cells at pH 7.8 (Figure 6). while both the fluorescence were negligible in cells at pH 7. This result suggested that enveloped pHs. K4L2ALA2K2LAKC. the nanoparticles were blocked to enter into the case of pH 6. Investigation of the drug loading capability and antitumor tively evaluate the amount of cellular uptake of free TPep(Rh B) and efficiency of the multifunctional enveloped MSN. While. This may be ascribed to the (Figure 3F). which was attributed was considered for leaking out of the mitochondrial matrix.8 (Figure 4D for 12 h and Figure 4E for 36 h.nature.6% by measuring the fluorescence emission of Furthermore. toxicity was not detected for cells treated with nanoparticles at pH the mitochondrial transmembrane potential (DYm) is an important 7. indicating that pH value has no influence on the cell (Figure 5A). JC-1 mainly accumulated in mitochon. To address the MSN-TPep(Rh B)/PEG-PLL(DMA) (Supplementary Figure S6). 48 h. unusual morphologies tumor cells with our enveloped MSNs was due to the TPT release and serious damage of mitochondria were observed. which would not release the therapeutic peptide before ility remained as high as 85% even at a concentration of 100 mg/L. the TPep(Rh cles. As shown in Figure pH 7. enveloped MSN.8% of cells remained survival with the concentration chondrial dysfunction.9. However. However. Thus. A scrambled peptide. While at pH 6.8 or the morphology of normal orthodox conformation in healthy cells. entering tumor cells. some of MSN-TPep/PEG-PLL(DMA) treated cells (11% of cells remained the mitochondrial membrane was broken (yellow doji star). the removal of the outer corona under acidic condition and discarding loading content of TPep(Rh B) in MSN-TPep(Rh B)/PEG. the disulfide bonds embedded accelerated TPT release was monitored with the treatment of GSH in the nanoplatform are able to respond to intracellular GSH for a at pH 6. Furthermore.4. while an increased amount of nanoparticles were targeting ability of TPP. morphological ility was lower than the corresponding non TPT loaded MSN evidence was further provided by Bio-TEM observation. also dependent cell uptake of the enveloped MSN-TPep/PEG-PLL(DMA) suggesting the the localization of nanoparticles in mitochondria. Thus.8 compared with that at (TPT@MSN-TPep(Rh B)/PEG-PLL(DMA)). the TPep moieties with the presence of GSH. On the to the additional inhibition of TPep. TPP was also conjugated to the control peptide.4 (Figure S9). similar low cytotoxicity were observed. Moreover. As shown in Figure S7. which demonstrated the layer was removed and improved cell lethality was observed for cells serious damage of mitochondria.4. at neously the decrease of J-aggre red fluorescence (normal mitochon. was used as a nega- B) release from MSN-TPep(Rh B)/PEG-PLL(DMA) was very slow tive control48. which survival at pH 6. the enveloped MSN-TPep(Rh B)/PEG. further suggesting a pH. the cell viability of KB cells treated with TPT@MSN-TPep*/ (control group. a number of black dots were found at the areas of mito- were also confirmed by Bio-TEM observation. To testify the advantage of co-delivery of a drug and therapeutic escence did not show significant difference to that of untreated cells peptide. in PLL(DMA)) at pH 7. Only 29. according to fluorescence measurement. For the cells treated with MSN-TPep*/PEG-PLL(DMA) nanocarrier offered a controlled release system for intracellular GSH (Figure 7B). Moreover. obvious cell cyto- with mitochondrial fluorescence probe JC-1 analysis. Due to the presence of outer protecting layer (PEG- fluorescent monomeric form remained in the cytoplasm.8). MSN-TPep/PEG-PLL(DMA) exhibited a indicator of mitochondrial functions. S8. a more significant antitumor activity was found in TPT@ ing (pentacle) and severe vacuolization (doji star). For KB cells (Figure 7B) at the same concentration. Whilst. The accel- evaluate mitochondrial death56. Thus.com/scientificreports The cell uptake of MSN-TPep/PEG-PLL(DMA) at different pHs other hand.6 mg/L of TPep).9. the collapse of DYm was observed by the gradually TPT@MSN-TPep/PEG-PLL(DMA) nanoparticles did not exhibit increased J-monomer green fluorescence in cytoplasm and simulta. after treating with MSN-TPep/PEG. In comparison with the above Figure 5B and 5C.4. was further loaded in the pores of the (Supplementary Figure S6A and S6B). tumor cells. 7. the viability of without treating with nanoparticles. Moreover. for nanoparticles incubated in the antitumor efficiency of the multifunctional enveloped nanoparti- absence of GSH or in the presence of GSH at pH 7. TPep and TPT co- SCIENTIFIC REPORTS | 4 : 6064 | DOI: 10. E).8 for 24 h.8. And the cell viab- To visualize the mitochondrial damage directly. As we know.8 (Figure 3G). due to the blocking effect of the outer shielding layer and the designated as TPep*. In addition. the change in DYm has been used to up to 100 mg/L (corresponding to . For TPep(Rh B) release study. the red fluorescence (TPep(Rh B)) and green fluorescence PLL(DMA) exhibited the ability of tumor-acidity-triggered targeting (TPT) intensity in KB cells at pH 6. results. an antineoplastic Free TPep(Rh B) gave a minimum cell uptake at both pHs drug topotecan (TPT). Thus. the green and red fluor. MSN-TPep/PEG-PLL(DMA) were incubated with KB cells PLL(DMA) was tested. Furthermore. about 2. for cells co-incubated with nanoparticles at pH the MSN-TPep/PEG-PLL(DMA). nanoparticles.4 for different time (Figure 4B and 4C). However.8 were dramatically enhanced. in vitro MTT assays were applied to explore the Rh B. there was a 3. For cells treated with nano. the cell viability of inefficient breakage of the disulfide bonds. which were identified to be the nanoparticles according to enon was observed. treated with TPT@MSN-TPep*/PEG-PLL(DMA). The drop of mitochondrial significant cytotoxicity to tumor cells after co-incubation for membrane potential is usually considered as the hallmark for mito. PEG-PLL(DMA) and TPT@MSN-TPep/PEG-PLL(DMA) were also dria to generate the red J-aggregates (J-aggre) along with slight green evaluated.1038/srep06064 5 . Figure 4A). most of the mitochondria showed irregular swell.4 (Figure 7D). and efficient delivering of TPep to tumor cell mitochondria. the cytotoxic effect of MSN-TPep/PEG- TPep.4. As shown in Figure 7C. As shown in Figure 7A.4 the shape and size (Figure 5D and 5E). the desired therapy to PLL(DMA) particles (Figure 5B. significant cytotoxicity to cells at pH 7.

environmental pH. the amide bonds can 10 mM. www. At could be successfully released from the nanoparticles due to the normal physiological pH.com/scientificreports Figure 4 | CLSM images with JC-1 assay of KB cells (A) without treatment. the surface charge of nanoparticles became reversed and the growth more efficiently with the synergistic effect. respectively.8 for 12 h and 36 h.3 nm were successfully prepared. Once arriving at tumor mitochondrial damage when KB cells were treated with the SCIENTIFIC REPORTS | 4 : 6064 | DOI: 10. Our results displayed that TPep and TPT PEG-PLL chains made the polymer become charge reversible.nature. of our nanocarriers. 2) a lipophilic TPP moiety for mitochondria therapeutic peptide (TPep) and PEG-PLL(DMA) on the surface of targeting. Following the programmed package of GSH triggered release. delivered by the enveloped MSN could suppress cancerous cell site. Well dispersed mesoporous silica nanoparticles with particle size of The therapeutic peptide TPep was composed of three parts: 1) a . indicating the good stability of the repulsion. we also demonstrated that such enveloped MSN pro. respectively. resulting in the removal in the absence of GSH. However. the surface charge of nanocarriers was sensitive to enveloped MSNs for storing drugs. It is well nanoparticles. much more nanoparticles could be internalized by cells at TPep/PEG-PLL(DMA)) was constructed for intracellular co. we detected serious stealth character during circulation in blood. which pro- tumor cells. indicating superior efficacy in delivering therapeutic agents delivery of two different therapeutic agents for synergistic therapy. (B) and (C) with MSN-TPep/PEG-PLL(DMA) treatment at pH 7. the amide bonds in PEG-PLL(DMA) were breakage of the disulfide bonds when the concentration of GSH is stable.8.1038/srep06064 6 . negligible TPep(Rh B) or TPT was released of stealth layer from the sheddable nanoparticles due to electrostatic from nanocarriers (Figure S10). vided the potential to develope GSH-sensitive nanodevices for select- itive to pH alteration. free cysteine residue which was favorable for anchoring onto mer- which presented the capability for encapsulating antitumor drug capto group functionalized MSN (MSN-SH) via disulfide bonds for TPT in their mesopores. (D) and (E) with MSN-TPep/PEG-PLL(DMA) treatment at pH 6. But at tumor acidic environment. Thus. even after 2 weeks’ incubation of the nanoparticles degrade to expose positively charged amines. nanoparticles transformed into a more cell-interactive form to dis- play enhanced interaction with tumor cells due to electrostatic attrac- Discussion tion. at pH 7.4. pH 6. Amides with b-carboxylic acid groups were highly sens.4 for 12 h and 36 h. a multifunctional enveloped nano-carrier (TPT@MSN.120 nm and pore diameter of . Compared to the cells incubated with enveloped nanoparticles In this study. known that the GSH concentration in bloodstream (2 mM in plasma) vided a strategy of targeted delivering pharmaceutical agents into and within cells (1–10 mM) is substantially different57. Scilicet. and 3) a cationic antibiotic peptide (KLAKLAK)2. The enveloped nanoparticles maintained their According to CLSM and bio-TEM images. the introduction of DMA moiety into ive release in living cells.

nature. The multifunctional enveloped MSN demonstrated here open a window for subcellular co-delivery of various therapeutic agents and will find great potential for tumor therapy. Tetraethylorthosilicatem (TEOS). To conclude. N-cetyltrimethylammonium bromide (CTAB). nanoparticles without compatible in vivo within desirable dose. and improved antitumor efficiency of the multifunctional enveloped MSN.8. In the cytotoxicity study. Although various bio-safety to induce cell apoptosis via the intrinsic mitochondria-dependent evaluations have provided a series of evidences that MSNs are bio- apoptosis pathway. hydrazine hydrate. many challenges that need to be overcome since the in vivo thera- introduction of TPep into the nanocarriers gave rise to a significant peutic use requires more strict performance modulation to satisfy the reduction in cell viability. complicated physiological environment so as to avoid unwanted bio- pores could further enhance the antitumor efficacy remarkably to performances of MSNs. there remain biocompatibility of the mesoporous silica nanoparticles. and they can be degraded therapeutic agents displayed negligible cytotoxicity. biological activities including biodegradability. excellent mito- chondria damage ability. and (D) enlarged rectangular area in C. In addition. including an obvious decline in mem. loading of TPT in the meso. The white arrows indicate the morphologies of mitochondria (M). (F) Energy-dispersive X-ray analysis of the selected area in D (red circle). biocompatibility. and ken). www. vacuolization. antineoplastic drug TPT co-delivered by the carrier could further inhibit the growth of tumor cells with remarkable synergistic effect. These data demonstrated that the released Tpep have the ablity bio-elimination of mesoporous silica.com/scientificreports Figure 5 | TEM images of (A) untreated KB cells (control group). enveloped MSNs at pH 6. Triphosgene was obtained SCIENTIFIC REPORTS | 4 : 6064 | DOI: 10. were purchased from Shanghai Reagent Chemical Co. Such multifunctional enveloped MSN exhibited ability of tumor acidic- triggered switching in surface charge. swollen.8. ninhydrin. and even bro. a multifunctional enveloped nano-carrier was con- structed by programmed packing mitochondria-targeted therapeutic agent and charge-conversional shielding layer onto MSNs. Rhodamine B. Methods Materials. and trifluoroacetic acid (TFA) Figure 6 | The control-release profiles of TPT at different conditions.1038/srep06064 7 . indicating good and expelled from the body via urine and feces58–61. However. In vitro results demonstrated a significant enhancement of tumor cellular uptake. (B) (C) and (E) KB cells treated with MSN-TPep/PEG-PLL(DMA) (50 mg/L) for 36 h at pH 6. realize the combined therapy with the synergistic effect. Moreover. It is worth noting that the multi-chemical modification could brane potential (DYm) and the apperance of unusaual mitochondrial tailor the surface property of MSNs. which in turn influence their morphology (cristae shrunk.

(4-Carboxybutyl) NaOH solution).36 mL TEOS was added dropwise to the modified poly(L-lysine) (PEG-PLL(DMA)). The resultant solution was poured into excess n-hexane to obtain Z-Lys-NCA. After completion of the reaction. and Dulbecco’s phosphate buffered saline (PBS) were obtained from Fmoc-protected amino acid was coupled on a 2-chlorotrityl chloride resin Invitrogen. Ninhydrin assay was utilized to monitor the coupling efficacy. Fluorescence analysis was performed pressure liquid chromatography (HPLC) with a C18 column using a liner gradient of on a RF-530/PC spectrofluorophotometer (Shimadzu).0 g. 2. FEI-QUANTA 200) and transmission electron microscopy (TEM.8 (100 mg/L of TPT@MSN-TPep/PEG-PLL(DMA) correspond to . was removed by dialyzation against water (the pH value was adjusted to 8. 7 mmol) were first dissolved in 480 mL of deionized water and the solution Synthesis of poly(ethylene glycol)-blocked-2.5-dimethylthiazol-2-yl)-2.19. www. Tetrahydrofuran (THF) was used under vacuum. Briefly.32 mmol/g) in a DMF solution of DiEA/HBTU/HOBt. 10 mL TFA was added to dissolve PEG-PLL(Z) (500 mg) in an diisopropylethylamine (DiEA). resuspended in distilled water and freeze-dried. Statistical significance in difference was analyzed using student’s T-Test: **p . 0. and Fmoc-Cys(Trt)-OH). The crude product was further recrystallixzed Surface modification of mesoporous silica nanoparticles. and was recrystallized before use. nanoparticles (500 mg) were first modified with (3- SCIENTIFIC REPORTS | 4 : 6064 | DOI: 10.N9-tetramethyluronium ice bath. Briefly. China). The modification of PEG-PLL (50 mg) hydrochloride.5%/2.nature. After stirring for another 2 h at 80uC. All the peptides were synthesized manually with fetal bovine serum (FBS). 1-hydroxybenzotriazole (HOBt). For synthesis of (KLAKLAK)2 sequence. Other reagents and solvents were of analytical grade and were purified (1.001. and dried under vacuum. (B) MSN-TPep*/PEG-PLL(DMA). All the (ASAP2020). N-fluorenyl-9. 0.32 mmol/g). After collecting and drying. to amino groups) was carried out in a NaOH solution (pH 8) tetraethylbenzimidazolycarbocyanine iodide) fluorescent dye. Perkin-Elmer). Brifly.1 g) was carried out in 15 mL Chlorotrityl chloride resin (100–200 mesh. topotecan and finally lyophilized to obtain PEG-PLL. After stirring for 15 min. loading: 1.6.N-dimethylformamide (DMF) at 50uC under N2 atmosphere for 3 days. nanoparticles were observed on scanning electron microscopy (SEM.39. 2.01 and ###p .4 and 6.1 M L-lysine (H-Lys(Z)-OH) were purchased from Sigma-Aldrich. o-benzotriazol-N. (Shanghai. and (D) TPT@MSN-TPep*/PEG-PLL(DMA) and TPT@MSN-TPep/PEG-PLL(DMA) at pH 7.59. dropwise to the suspension and stired for 3 h. Synthesis of silica nanoparticles. followed by precipitation in cold ether. 2 eq. Characterizations. the resin was finally permeation chromatographic (GPC) system consisting of Waters 2690D separations washed with DMF (four times) and dichloromethane (DCM) (four times) and dried module and Waters 2410 refractive index detector. penicillin-streptomycin. Synthesis of peptide analogs.74 mmol) and NaOH (0. 3-(4.28 g. H-Lys(Z)-OH (7. The final product was obtained by lyophilization. The mixture was precipitated in an excess of diethyl ether three times to obtain PEG- Ala-OH. PLL(Z). The polymerization of Z.4 g) in THF (10 mL) was added and methanol. The morphologies of acetonitrile and deionized water containing 0.0 by 0. Nitrogen (95%/2. The mitochondria fluorescence probe (Mito Tracker Green FM). anhydrous N. and N(e)-benzyloxycarebonyl. 1H NMR spectra were recorded on a Varian Unity 300 MHz D-amino acids were used for avoiding proteolysis62.5%). Mw 2000). was removed with 20% piperidine in DMF (v/v) for twice. The particle size and zeta potential were expected samples from the dried resin were performed by a mixture of TFA/TIS/H2O measured using Malvern Zetasizer Nano-ZS ZEN3600.5-diphenyltetrazolium standard solid phase synthesis protocols based on classical Fmoc-chemistry. Then. HBr (5 mL of a 33 wt% solution in acetic acid) was hexafluorophosphate (HBTU). the product was resuspended in 10 mL DMF 2.3.3 mL/min.01 and ***p . methoxycarbonyl (FMOC) protected amino acids (Fmoc-D-Lys(Boc)-OH.0 g) by amino-terminated PEG (0. CTAB (1.1038/srep06064 8 . (C) MSN-TPep/PEG-PLL(DMA). with DMA (75 mg.2. 0.6 g) was suspension under vigorous stirring. After the completion of the synthesis. Fully removal of side chain protected groups and cleavage of the as the eluent at a flow rate of 0. vacuum dried. JC-1 (5. from Shanghai Chemical Reagent Co.3-dimethylmaleic anhydride. 5. Then a solution of triphosgene (3.com/scientificreports Figure 7 | Cell viability of KB cells incubated with (A) free TPep and TPep*. for 24 h under N2 atmosphere.N9. ## p .1% TFA. The crude product was adsorption2desorption isotherms were measured on a micromeritics instrument collected. washed thoroughly with deionized water atmosphere. Fmoc-D-Leu-OH. The above obtained silica from dried THF/n-hexane twice and dried under vacuum.69-tetrachloro-1.001. bromide (MTT). diethy ether. triphenylphosphonium bromide was purchased from Adamas Reagent Co. piperidine. Then the solution was precipitated in triisopropylsilane (TIS) were purchased from GL Biochem Ltd.3-Dimethylmaleic anhydride (DMA) and (3-mercaptopropyl)trimethoxysilane and the solution was dialyzed (MWCO: 3500 Da) against distilled water for 3 days were purchased from Aladdin Reagent Co. Fmoc-D. Thermal gravitational analysis (TGA) was performed with a Thermo samples listed in Table S1 were synthesized and the purity was examined by high- Gravimetric Analyzer (TGS--II. The triphenylphosphonium bromide and Rhodamine B was coupled using the similar molecular weight and polydispersity index (PDI) of PEG-PLL were evaluated by gel method to amino acid. Ltd. was stirred at 80uC for 15 min. Fmoc-protecting group before use. 0. Ltd. RPMI-1640 Medium. (4-Carboxybutyl) spectrometer by using dimethyl sulfoxide-d6 (DMSO-d6) or D2O as the solvent.65 mg/L of TPT). the excess of DMA monomethoxypoly(ethylene glyco) (PEG. Lys-NCA (1. Fmoc-Lys(Dde)-OH. JEM-2100).N. Subsequently. the dissolved in distilled THF (80 mL) and the mixture was stired at 50uC under N2 resulting white precipitate was centrifuged. amino. and added dropwise and was stirred for another 1 h.

132.. The solution was put into a Ed. 140–146 (2011).. E. D. Co-delivery of drugs and and washing with PBS. fluorescence pharmaceutical applications. 792–801 (2014). Finally. Poly(ethylene glycol) in for Tpep(Rh B) release study and TPT@MSN-Tpep/PEG-PLL(DMA) for TPT release drug delivery: pros and cons as well as potential alternatives. Wang. 23. Wang. & Nie. van der Aa. respectively. At designated time intervals. et al. Subsequently. study. Magnetic nanoparticles with dual functional properties: drug Topotecan (TPT) loading. www. U. Yamada. et al. Enhanced intracellular delivery of quantum dot respective standard curve. & Srivastava. CTAB was removed by refluxing the nanoparticles (500 mg) containing RPMI-1640 media (200 mL). Y. M.8. et al. After removing the medium and washing with PBS. C. Int. Engineering the assemblies of biomaterial medium containing nanoparticles (30 mg/L) was added and the cells were incubatied nanocarriers for delivery of multiple theranostic agents with enhanced antitumor for 4 h at 37uC. the followed by adding PEG-PLL(DMA) (6 mg) and stirring for 4 h to obtain MSN. MSN-TPep/PEG-PLL(DMA) 18. H. All the cells were digested by trypsin. Mat. M. 994–1005 (2013). Codelivery of an optimal drug/siRNA combination using and cultured in RPMI-1640 medium (1 mL) containing 10% FBS and 1% antibiotics mesoporous silica nanoparticles to overcome drug resistance in breast cancer in for 24 h.. Drug Discov. T. & Yang. The dissolution was subsequently analyzed by RF-5301PC 7.com/scientificreports mercaptopropyl)trimethoxysilane (5 mL) in 30 mL methanol upon stirring at room In vitro cytotoxicity. & Schubert. Surface charge conversion of nanoparticles at different pHs.4.8 with 1 M HCl. Shen. Yang. Y. free TPep(Rh B) (5 mg/L) and MSN-TPep(Rh B)/PEG. H. Bioconjug. 1.000 U/mL) and incubated in a 14. Proteomic analysis of cancer- cells were filtrated and examined by flow cytometry (BD FACSAriaTM III). KB cells were seeded in a 96-well plate (1. BD Laser).. after co-incubation of KB cells with 391–411 (2013). Q.4% HCl (6 mL) for 24 h at 60uC to obtain 5% CO2).1038/srep06064 9 . Nakamura. Mater.0). H. Hiraku. in 0. Then the medium was removed and nanocapsules for intracellular cancer drug delivery.1 M PBS) at 4uC overnight. Richard. He. 771–781 (2012). 4259–4265 (2010). Angew. MSN-SH particles (300 mg) were dispersed in 30 mL particular materials (free TPep*. Gao.789–795 (2013). Prodrugs forming high drug loading multifunctional were further incubated at 37uC for another 4 h. Chem. Gold nanoparticles in PLL(DMA) nanoparticles were incubated in 10 mM PBS at a pH of 7.nature. Rev. 9335–9346 (2012). G. H. the medium room temperature for 24 h to obtain the desired MSN-TPep. The optical density (OD) was TPep/PEG-PLL(DMA). 1113–1121 (2012). For and camptothecin-resistant variants. After particular time intervals. et al.. Verma. Sheddable ternary nanoparticles for tumor acidity-targeted spectrofluorophotometer (lex 5 488 nm for TPep(Rh B). G. & Kawanishi. Drug Deliv. Soc. Meng. & Montaudon.4 with 0. & Harashima. Mok. was added to each well and the cells were further incubated for 4 h. (50 mg/L) dispersed in RPMI-1640 medium (1 mL) were added and the cells were Mechanism of apoptosis induced by doxorubicin through the generation of further incubated at 37uC for another 4 h. Cells cell mitochondria. Tarn. After removing the medium 19. K. 26. Chem. Cheng. & Park. In vivo treatment of tumors using host-guest conjugated (50 mg/L) dispersed in RPMI-1640 medium (1 mL) were added and the cells were nanoparticles functionalized with doxorubicin and therapeutic gene pTRAIL. washed with methanol or PBS (10 mM. The mitochondria were stained with JC-1 Biomaterials 33. and 1% antibiotics (penicillin-streptomycin. 45. Adv. Z. A. observed under a confocal microscopy. 46. P. and PBS with 10 mM GSH at pH 6. J. V.5 3 104 cells/well) temperature overnight. Y. Y. Each vacuum. 5329. T. M. Yang. K. C. study) were suspended in 2 mL PBS buffer solution. Sidransky. and MSN-TPep(Rh B)/PEG-PLL(DMA) 15. J.. Mater.. M. In vitro release experiments were carried out in four different 8. the cells were observed under a confocal microscopy. Am. Core-shell nanosized assemblies mediated by the a-b above. 25. Nat. Drug Deliv. Rev.. et al. 6288–6308 (2010). vitro and in vivo. C. Chem. further incubated at 37uC for 12 h or 36 h. Robert. Zhang. Adv. Kojima. SCIENTIFIC REPORTS | 4 : 6064 | DOI: 10. 3. 49. Biomaterial 29. DNA from cationic core–shell nanoparticles self-assembled from a biodegradable copolymer. De. pH 8. Res. V. Sheddable ternary nanoparticles for tumor acidity-targeted cells) were seeded in a glass bottom dish in RPMI-1640 medium (1 mL) with 10% FBS siRNA delivery. The suspended 22. completely dissolving of 1 mg TPT@MSN-TPep/PEG-PLL(DMA) in 0. pH 8. Angew. Int. concentration of 1 mg/mL at 37uC. Kagan. et al. et al. S. S. 50 mg of the above TPep/PEG-PLL(DMA)) at different doses. Ye. nanoparticles. topotecan hydrochloride solution (1 mg/mL) in PBS (10 mM. MSN-TPep*/PEG-PLL(DMA). 2741–2750 (2013). 1439–1451 (2005). Bio-TEM observation. Br. H. Lipid-peptide vesicle nanoscale hybrids for triggered drug solution. zeta-potentials were measured by Malvern Zetasizer Nano-ZS ZEN3600.. ACS Nano 7. Y. M. E. without treatment were used as control. The programmed packing of TPT-loaded 321–324 (2009). R. H. and adenovirus nanoparticles triggered by acidic pH via surface charge reversal. Nagasaki. ACS Nano 6. Kherfellah. J.. A spectrofluorophotometer. J. Wu. Tada-Oikawa. T. Subsequently. 64. J. S. Flow cytometry analysis.. T. PBS without multifunctional mitochondria-targeted agents in cancer therapy. the cells were collected. For JC-1 assay.. Z. Han.4. Then. lex 5 350 nm for TPT). et al. 1307–13015 (2008). Kim. the cells were combination of camptothecin and doxorubicin or etoposide on rat glioma cells observed under a confocal microscopy (CLSM. Cancer 85. ZnO nanoparticles applied to bioimaging and drug delivery. the culture media was replaced with 200 mL medium containing mesoporous MSN-SH. Y. Yoon. in a mixture of methanol (100 mL) and 37.. W. Confocal laser scanning microscopy. H..4) and stirred at 4. carefully washed with PBS three times. 797–801 (2008).. Res. Al-Ahmady. The solution was TPep/PEG-PLL(DMA). Acc. medium was removed and DMSO (200 mL) was added. X. the 2. 5 mg of the corresponding nanoparticles (MSN-Tpep(Rh B)/PEG-PLL(DMA) 9. S. The unclear pore complex: the gateway to successful humidified atmosphere containing 5% CO2 at 37uC for 24 h. 791–796 (2006). the cells were washed three times with PBS. H. 1 mL of RPMI-1640 17. solution. D. MSN-SH with TPep and PEG-PLL(DMA) were similar to the procedure described 5. 60. KB cells (human mouth epidermal carcinoma 13. PLL(DMA) (50 mg/L) dispersed in DMEM medium (1 mL) were added and the cells 21. B. After 4211–4219 (2010). & Kataoka. H. Quan. G. 4012–4021 (2008). Mater. Cancer 3.–5335 (2013). Versatile fluorescence resonance epoxy resin and sliced with a thickness of 50–70 nm. D.. The pH of the incubation medium in some resulting material and TPep (50 mg) were dispersed in 30 mL DMF and stirred at wells was adjusted to 6. Park. Y. embedded in 25. MSN- methanol solution containing 2. Rev. The nanoparticles were isolated by centrifugation and magnetic resonance imaging and drug delivery. After removing the medium and washing with PBS. ACS Nano 7. In vitro release study.0). 50 mg of MSN-TPep was replaced with 200 mL of fresh medium. Fischer. Mater. & Lee. Pavillard.. For cell uptake nonviral gene delivery. Hollow manganese oxide nanoparticles as multifunctional agents for room temperature overnight. and TPT@MSN- further stirred at room temperature overnight. and value was averaged from four independent experiments. et al. Then.8 with a delivery applications. Zhang. Life Sci.. KB cells were seeded in 6-well plates (5 3 104 cells/well) 20. Y. Chem. The cumulative release of TPT or TPep(Rh B) was calculated according to the 12. Shah. and dried under viability was calculated as: cell viability (%) 5 (ODtreated/ODcontrol) 3 100%. ACS Nano 6. & Shi. MSN-TPep/PEG.. Mesoporous silica nanoparticle nanocarriers: biofunctionality and nanoparticles and fixed with 1 mL general fixative (containing 2. Z.7. collected. S. Hoogenboom. Adv.1 M NaOH release by mild hyperthermia in vitro and in vivo. Mizutani. 100 mg of MSN-SH were suspended in 15 mL of delivery and magnetic resonance imaging. Hatakeyama. MSN-TPep(Rh B)/PEG-PLL(DMA). Ed. Ghosh. MSN-TPep*/PEG-PLL(DMA). The resulting TPT@MSN-TPep/PEG-PLL(DMA) nanoparticles were cyclodextrin dimer with a tumor-triggered targeting property. Shin. respectively. ACS Nano 6. the mitochondria were hydrogen peroxide.5% glutaraldehyde biocompatibility.. PBS with 10 mM GSH at pH 7. the pH value of the dissolution was adjusted to . Su. TPT@MSN-TPep*/PEG-PLL(DMA). evaluation. stained with 100 nM Mito Tracker Green FM in RPMI-1640 medium (without FBS) 16. free TPep(Rh B) (5 mg/L). Subsequently. Adv.. the cells were washed with PBS for 3 times to remove excess 24. the particles were isolated measured at 570 nm with a microplate reader (BIO-RAD 550). Jain. Otsuka. Res. For TEM observation. Then.1 M HF 6.8. and washed twice with PBS (pH 7.. siRNA delivery. et al. free TPep. After each functionalization step. Acc. B. of drug release. Y.. H. The relative cell by centrifugation. D. the cells were efficacy. dialysis tube (MWCO: 12 kDa) which was directly immersed into 10 mL incubation 10. Pharm. S. 1616–1622 (2013). 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