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Taming the Wildness of “Trojan-Horse” Peptides by Charge-Guided Masking and Protease-Triggered Demasking for the Controlled Delivery of Antitumor Agents Nian-Qiu Shi†,‡ and Xian-Rong Qi*,†,§ †
Beijing Key Laboratory of Molecular Pharmaceutics and New Drug Delivery System, School of Pharmaceutical Sciences, Peking University, Beijing 100191, P. R. China ‡ School of Pharmacy, Jilin Medical University, Jilin City 132013, P. R. China § State Key Laboratory of Natural and Biomimetic Drugs, Beijing, 100191 P. R. China S Supporting Information *
ABSTRACT: Cell-penetrating peptide (CPP), also called “Trojan Horse” peptide, has become a successful approach to deliver various payloads into cells for achieving the intracellular access. However, the “Trojan Horse” peptide is too wild, not just to “Troy”, but rather widely distributed in the body. Thus, there is an urgent need to tame the wildness of “Trojan Horse” peptide for targeted delivery of antineoplastic agents to the tumor site. To achieve this goal, we exploit a masked CPPdoxorubicin conjugate platform for targeted delivery of chemotherapeutic drugs using charge-guided masking and protease-triggered demasking strategies. In this platform, the cell-penetrating function of the positively CPP (D-form nonaarginine) is abrogated by a negatively shielding peptide (masked CPP), and between them is a cleavable substrate peptide by the protease (MMP-2/9). Protease-triggered demasking would occur when the masked CPP reached the MMP-2/9-riched tumor. The CPP-doxorubicin conjugate (CPP-Dox) and the masked CPP-Dox conjugate (mCPP-Dox) were used as models for the evaluation of masking and demasking processes. It was found that exogenous MMP-2/9 could effectively trigger the reversion of CPP-cargo in this conjugate, and this trigger adhered to the Michaelis−Menten kinetics profile. This conjugate was sensitive to the trigger of endogenous MMP-2/9 and could induce enhanced cytotoxicity toward MMP-2/9-rich tumor cells. In vivo antitumor efficacy revealed that this masked conjugate had considerable antitumor activity and could inhibit the tumor growth at a higher level relative to CPP-cargo. Low toxicity in vivo showed the noticeably decreased wildness of this conjugate toward normal tissues and more controllable entry of antitumor agents into “Troy”. On the basis of analyses in vitro and in vivo, this mCPP-cargo conjugate delivery system held an improved selectivity toward MMP-2/9-rich tumors and would be a promising strategy for tumor-targeted treatment. KEYWORDS: “Trojan-Horse” peptides, taming the wildness, charge-guided masking, protease-triggered demasking, controlled delivery
1. INTRODUCTION
However, another crucial characteristic of CPP is the noticeable cellular ubiquity of its transduction ability.16,2 As every coin has two sides, the nonselectivity of CPP plays dual roles in the targeted cargo delivery for tumor therapy.2 For one thing, CPP could potentially translocate any cells and tumor tissues barriers in theory due to its nonselectivity,2 without involving any distinctive biomarkers (e.g., receptors) commonly used in traditional passive or active targeting notions.17,2 For another thing, the risk of off-target toxicity or increased adverse
Cell-penetrating peptide (CPP), often vividly named as the “Trojan Horse” peptide, is a sort of short peptide that generally consists of no more than 30 amino acids.1,2 CPP presents a outstanding profile to transport hugely diverse payloads from perspectives of size and biological nature.3−5 Noticeably, there are numerous clinical or preclinical evaluations of CPP-cargo conjugates, demonstrating obvious advantage as therapeutics in the field of drug discovery.2,6 Conjugation of CPP to antitumor drug has been demonstrated as an attractive approach to overcome multidrug resistance and achieve an improved therapeutic efficacy for a series of tumors.7−15 © XXXX American Chemical Society
Received: January 21, 2017 Accepted: March 7, 2017
A
DOI: 10.1021/acsami.7b01056 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
Figure 1. Taming schematic diagram against the wildness of “Trojan-Horse” peptide by means of charge-guided masking and protease-triggered demasking strategy, and proposed controlled delivery of antitumor agents to “Troy” in the designed masked CPP-cargo conjugate. The masked CPPcargo conjugate in circulation did not show the cell-penetrating function, due to the positively charged CPP was masked by intramolecular electrostatic interactions of a negatively shielding peptide by a substrate peptide linker cleaved by the trigger of a protease (MMP-2/9). Once the masked CPP-cargo conjugate got into the tumor site, it can be unsealed by the overexpressed extracellular MMP-2/9 reversed to CPP-cargo conjugate with the wildness, and then carry chemotherapeutical cargo to pierce through the membrane barrier for the tumor therapy, and the controlled delivery of CPP-cargo to “Troy” would be achieved.
peptide that can be cleaved by protease is used to connect the two sections. It is known that proteases, such as matrix metalloproteinase 2 and 9 (MMP-2/9), are overexpressed in many stages of cancers.25 In normal (nontarget) tissues, CPP is sealed to the masked CPP form by the charge-guided masking. Once it reaches the tumor tissue (target site), the masked CPP is unsealed into the CPP form by a protease-triggered demasking strategy. Subsequently, the CPP can bring cargo enter the tumor cells to play a therapeutic role (Figure 1). In this work, we constructed CPP-doxorubicin (CPP-Dox) and masked CPP-doxorubicin (mCPP-Dox) conjugates. The protease-triggered demasking feature of mCPP-Dox was investigated for understanding the taming profile of masking/ demasking strategy on the wildness of “Trojan Horse” peptides. The efficacy and side effects of antitumor treatment were studied in xenografts-bearing BALB/c nude mice for the evaluation of in vivo selectivity of the platform based on masking/demasking strategies. The works provide a new perspective to overcome the wildness of “Trojan Horse” peptides and control the delivery of antitumor agents to “Troy”.
reaction would increase in the light of the nonselective uptake profile of CPP-cargo.2 So, an intractable problem, i.e., “Is the “Trojan Horse” peptide too wild to go only to “Troy”?” was put forward by Eric Vives.16,2 Strengths of CPP in targeting delivery might be cut down by the nonspecific distribution found in some publications, due to the fact that CPP could cause a marked nonselective combination through guiding linked cargoes toward normal tissues or cells.18,19,2 It is pressing to tame the wildness of “Trojan Horse” peptide, and make the peptide go only to “Troy” by enhancing their selectivity in order to avoid probable unbearable adverse reaction toward health cells or tissues. Noticeably, the wildness of “Trojan Horse” peptide, has become a troubled problem when it is exploited to deliver antitumor drugs in vivo after the administration for the targeting treatment.20,21 To tame the wildness of “Trojan Horse” peptide, we employ charge-guided masking and protease-triggered demasking strategy to yield an “off−on” switch of CPP function for the targeted delivery of antitumor chemotherapeutical drugs.5,22−24 In this system, the positive charge of CPP is masked by appending negatively charged shielding peptide. A substrate B
DOI: 10.1021/acsami.7b01056 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
2.4. Secondary Structure Assay. Secondary structures of mCPP and mCPP-Dox conjugates were studied by analyzing ellipticity spectra from 190 to 500 nm utilizing a circular dichroism (CD) spectrometer (J-810, Jasco, Japan). The mCPP or mCPP-Dox was dissolved at 0.1% (1 mg/mL) in deionized water. These solutions were then added into a cuvette with 1 mm path length for obtaining spectra. The data were calculated according to the formula: [θ]λ = θobs × 1/(10Lcn), where [θ]λ = molar ellipticity at λ in deg cm2 d/mol, θobs = observed ellipticity at λ in mdeg, L = path length in cm, c = concentration of peptide in mol/L, and n = number of amino acids in the peptide. Spatial structure of mCPP was further simulated by using the Discover 3.0 software package in Insight II. 2.5. Exogenous MMP-2/9-Triggered Demasking Characteristics Assay. Exogenous MMP-2/9-triggered demasking characteristics were evaluated by enzymatic cleavage experiment. This performance was run a seal jar in a water bath at 37 °C. Activated MMP-2/9 (collagenase IV) was achieved by adding the 2.5 mM 4aminophenylmercuric acetate solution at 37 °C for 1 h. The 0.5 mL of activated MMP-2/9 was injected into 0.5 mL of mCPP-Dox solution. During this period, aliquots of the mCPP-dox and MMP-2/9 mixture were collected at different time ponits (1, 2, 3, and 4 h). The content of conjugate was determined in a HPLC system containing a Waters 2487 dual wavelength Absorbance Detector and 1525 pump at 220 nm. The flow was 1 mL/min. A mixed mobile phase was applied including solvent A (H2O with 0.1% TFA) and solvent B (acetonitrile with 0.1% TFA). The gradient elution was exploited: 0 min-15 min, from 95% solvent A to 70% solvent A; 15 min to 25 min, from 70% solvent A to 100% solvent B. 100% solvent B was maintained for 5 min in this system. 2.6. Endogenous MMP-2/9-Controlled Cells Antiproliferative Assay. Endogenous MMP-2/9-controlled cells antiproliferative activity was assessed by MTT method. The antiproliferative activity of parent drug (Dox), mCPP-Dox, CPP-Dox, and carriers (CPP and mCPP) was measured. HT-1080 and MCF-7 cells were cultured in 96well plates under a seeding density of 2 × 104 cells/well. In an incubator (5% CO2, 37 °C), cells grew for another 24 h. After that, 20 μL of samples with various concentrations ranging from 0.05 μM to 100 μM were put into each well, and were treated for different time (24 or 48 h). After incubation, each well was mixed with 5.0 mg/mL of MTT solution (20 μL). And all samples were kept at 37 °C for 4 h. Supernatant in each well was disgarded and 200 μL of DMSO was added. The plate was rocked at a shaker to dissolve generated dark crystals. By a microplate reader (Bio-Rad, U.S.A.), the optical density (OD) determination was performed at 570 nm. The cell viability = B/ A × 100%. In this formula, “A” represents the OD value yielded from an incubation of the cells and the culture medium, and “B” represents the OD value yielded from an incubation of the cells and samples. All measurements were assessed in sextuplicate. 2.7. In Vivo Antitumor Efficacies. About two million HT-1080 cells were inoculated subcutaneously into the armpits of the BALB/c nude mice. There were three therapeutic schedules (I, II, and III) for the experiment when xenografts reached to approximately 50 mm3 in volume. Schedule I started on day 5 after tumor inoculation. Animals were divided into six groups randomly (seven animals for every group) and were treated with free Dox (2 mg/kg), CPP-Dox (2 mg/kg Dox equivalents), mCPP-Dox (2 mg/kg, 5 mg/kg, and 10 mg/kg Dox equivalents) and physiological saline via the tail vein on day 5, day 8, and day 11 after tumor inoculation for three times. Schedule II started on day 9 after tumor inoculation. Animals were divided into three groups randomly (nine to ten animals for every group) and were treated with physiological saline, free Dox (5 mg/kg) and mCPP-Dox (5 mg/kg Dox equivalents), respectively, via the tail vein on day 9, day 12, and day 15 after tumor inoculation for three times. Schedule III started on day 6 after tumor inoculation. Animals were divided into five groups randomly (six animals for every group) and were administrated with Control (physiological saline), free Dox (5 mg/kg, 10 mg/kg, and 15 mg/kg) and mCPP-Dox (15 mg/kg Dox equivalents) via the tail vein on day 6, day 9, and day 12 after tumor inoculation for three times.
2. EXPERIMENTAL SECTION 2.1. Materials. Doxorubicin hydrochloride (>99%) was supplied by Hisun Pharmaceutical Co., Ltd. (Zhejiang, China). N-Succinimidyl 3-maleimidopropionate (SMP) (more than 98%) was provided by Jiaxing Biomatrix Co., Ltd. (Zhejiang, China). Trypsin was purchased from Amresco Inc. (Ohio, U.S.A.). 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide and collagenase IV were provided from Sigma-Aldrich (St. Louis, U.S.A.). Penicillin, streptomycin, and Hoechst 33258 were supplied by Macgene Co. Ltd. (Beijing, China). All other chemicals were of analytic grade or HPLC grade. The CPP (LGA-rrrrrrrrrC) and masked CPP (mCPP, DGGDGGDGGDG-PLG*LAG-rrrrrrrrrC) were prepared based on a standard Fmoc solid phase peptide synthesis approach by Shanghai GL Biochem Co. Ltd. (Shanghai, China). L-Amino acids can be indicated by capital letters and D-amino acids can be indicated by lowercase letters. The purity of mCPP and CPP was above 95%. 4Aminophenylmercuric acetate was achieved from Merck Co. Ltd. (Darmstadt, Germany). 2.2. Cell Lines and Animals. Human fibrosarcoma HT-1080 cells were afforded from Cell Culture Centre, Peking Union Medical College (Beijing, China). The cells grew in culture media containing Minimum Essential Medium with Earle’s salts, L-glutamine, nonessential amino acid (Macgene, Beijing, China), 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 μg/mL streptomycin. Human breast adenocarcinoma MCF-7 cells were obtained from the Institute of Hematology & Blood Diseases Hospital (Tianjin, China). The cells were cultured in Dulbecco’s Modification of Eagle’s Medium with glucose, sodium pyruvate, L-glutamine, and HEPES (Macgene, Beijing, China) added by 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin.22 An incubator operated at 37 °C under 5% CO2 at fully humidified conditions was used to culture these cells. All assays were carried out when cells grew in the exponential growth phase. BALB/c nude mice (5−8 week-old, 15−20g body weight) were obtained from the Department of Experimental Animals, Peking University and maintained under standard housing conditions. All of the animal experiments adhered to the principles of care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee of Peking University. 2.3. Synthesis and Characterization. SMP, Dox, and triethylamine were dissolved in dimethylformamide with a molar ratio of 1:1.1:2 accompanied by continuous stirring for 2 h at room temperature. The reaction process was monitored by thin-layer chromatography (TLC) using a mixed expanding agent (methanol/ chloroform/ammonia =3:7:0.3, v/v). Cold anhydrous diethyl ether was added into reactive solutions to recrystallize the product. The precipitate was washed using anhydrous diethyl ether and collected through centrifugation procedure. The red product was dried at a vacuum condition for 24 h. Thus, N-succinimidyl 3-maleimidopropionate-Dox (SMP-Dox) derivative designed as a linker was achieved for the further coupling of required conjugates. CPP-Dox and mCPP-Dox conjugates were synthesized by a Michael addition reaction between thiol group in peptides and maleimide group in SMP-Dox derivative.5 Briefly, peptides (CPP or mCPP), SMP-Dox derivative, and triethylamine were dispersed in dimethylformamide at a molar ratio of 1:1:21, and subjected to roomtemperature mixing for 2 h. Above TLC condition was used to detect the reaction process. Cold anhydrous diethyl ether was added into reactive solutions to recrystallize these products. The products were washed by anhydrous diethyl ether and collected by centrifugation. Matrix-assisted laser desorption-mass spectrometry (MALDI-TOFMS) was used to examine the molecular weight of products. The content was also measured by high-performance liquid chromatography (HPLC). A Waters 2487 containing Dual Wavelength Absorbance Detector and 1525 Pump (Milford, MA) was adopted as a HPLC system, and was operated at a wavelength of 254 nm. A solvent (H2O containing 0.1% trifluoroaceticacid [TFA]) mixed with another solvent (acetonitrile containing 0.1% TFA) at a ratio of 30:70 (v/v) was employed as a mobile phase running at a flow rate of 1 mL/ min. C
DOI: 10.1021/acsami.7b01056 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
Figure 2. Structure and synthesis scheme of CPP-Dox or mCPP-Dox conjugates based on active ester reaction and Michael addition reaction. (A) Dox, (B) N-succinimidyl 3-maleimidopropionate (SMP), a linker to link CPP or mCPP with Dox, (C) SMP-Dox derivative, (D) CPP-Dox, and (E) masked CPP-Dox conjugate.
penetration and the reality of undesired drug exposure. “Trojan Horse” peptide is popularly powerful arm like “spear” to break through membrane barriers like “shield” for the delivery of antitumor drug.26 CPP facilitates the delivery of cargoes across the impermeable barriers, but it is too wild and also brings about an indiscriminate internalization into all kinds of cells/ tissues.4,18,19,26 An ideal drug delivery system should exert its specific action at the desired targeted region and avoid the exposure toward the health tissues. Thus, the “Trojan Horse” must be controlled and their wildness should be tamed for mediating the delivery of antitumor drug to achieve both therapeutic efficacy and safety.2,26 In this study, we employ the charge-guided masking approach to tame the wildness of “Trojan Horse” peptide and adopt the protease-triggered demasking strategy to regain its penetration ability. In designed mCPP-Dox conjugate (DGGDGGDGGDGPLG*LAG-rrrrrrrrrC−Dox, as shown in Figure 2), there are five units: (i) anticancer drug Dox; (ii) a linker for conjugating CPP to Dox; (iii) CPP, a positively charged D-form nonaargigine (r9); (iv) a substrate peptide PLA*LAG cleaved by the trigger of MMP-2/9 to demask the shielding peptide; and (v) a negatively charged shielding peptide GDGGDGGDGGD for masking CPP. A detailed delivery process was depicted in Figure 1. Here, in terms of high transduction efficiency and enhanced metabolic stability, we use synthetic D-form nonaarginine as a typical CPP instead of naturally originated CPPs. Non-natural D-form polyarginine
Two indicators including tumor volumes and body weights were monitored every day. The tumor volume = π/6 × A × B2, where A was the long diameter, and B was the short diameter of xenografts measured with vernier calipers. After approximately 15 days chemotherapy, animals were euthanized and considered as dead. Xenografts were separated from animals and weighed. 2.8. In Vivo Toxicity Evaluation. In vivo toxicity study was investigated by body weight changes, animal survival, hemogram analysis, and tissue section during or after the period of antitumor activity. Blood was taken from the retro-orbital venous plexus on the day 21 (five animals for each group) during the therapeutic schedule II, and on the day 10, day 19 (four to five animals for each group) during the therapeutic schedule III. Hematological indicators including red blood cell (RBC), hemoglobin (HGB), white blood cell (WBC), and platelet (PLT) were determined with a hematology apparatus (MEK-6318K, Japan). Tissues (heart, liver, spleen, lung, kidney, and intestine) of sacrificed animals in the therapeutic schedule II and III were fixed in 10% buffered formalin overnight. Paraffin embedding was carried out and tissue sections were prepared based on standard histological protocols. For further morphologic observation, tissue sections were treated by a staining procedure using hematoxylin and eosin (H&E). 2.9. Data Analysis. Data were presented as mean ± standard deviation and statistical analysis was carried out by unpaired Student’s t test. Data were considered significantly different at p < 0.05.
3. RESULTS AND DISCUSSION 3.1. “Taming” Strategy, Synthesis, and Characterization. In the field of drug delivery, researchers were often subjected to the dilemma between the requirement of drug D
DOI: 10.1021/acsami.7b01056 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
Figure 3. (A) CD spectra of mCPP and mCPP-Dox; (B) half-time of triggered cleavage of mCPP-Dox conjugate in different ratio of triggered enzyme (MMP-2/9)/mCPP-Dox (w/w); (C) Michaelis−Menten curve fitting of MMP-2/9-triggered cleavage kinetics characteristics of mCPP-Dox. (D) IC50 values of mCPP-Dox, CPP-Dox, and Dox was determined using the MTT assay in HT-1080 and MCF-7 cells after 24 and 48 h incubation. IC50 ratios (HT-1080/MCF-7) of mCPP-Dox, CPP-Dox, and Dox are shown in the inset.
+K]+): 733.6 (theoretical), 733.7 (practical) (Figure S1 of the Supporting Information (SI)); HPLC analysis: 94.6% (Retention time is 38.986 min) (Figure S2). CPP-Dox: MALDITOF-MS (m/z, [M + H]+): 2462.78 (theoretical), 2463.8 (practical) (Figure S3); HPLC analysis: 95.8% (Retention time is 3.866 min) (Figure S4); Yield: 60.4%. mCPP-Dox conjugate: MALDI-TOF-MS (m/z, [M−H]+): 3588.8 (theoretical), 3587.26 (practical) (Figure S5);2 HPLC analysis: 95.3% (Retention time is 4.573 min) (Figure S6); Yield 78.8%. On the basis of the reasonable conjugation method and characterization from data, CPP-Dox and mCPP-Dox were obtained successfully. 3.2. Beta-Sheet Secondary Structure before Cleavage. The spatial structure of the mCPP-Dox is important to induce its cleavage triggered by MMP-2/9 before its arrival at the tumor tissue. Polypeptide or biomacromolecule tends to form secondary structure or tertiary structure. The tertiary structure of some macromolecules has more complicated steric hindrance and is unfavorable for their close to the trigger of protease. The secondary structure of macromolecules can be reflected by the CD data in the far UV region ranging from 180 to 250 nm. The side chain tertiary structure of macromolecules can be deduced based on CD data in the near UV region ranging from 250 to 350 nm. A minimum value at 216 nm and a maximum value at 195 nm in CD spectra were a typical profile for beta-sheet structure. The structure of random coils
exhibited the most excellent transduction efficiency relative to other CPPs.27 Metabolic stability of CPP increased significantly when non-natural D-amino acids replaced natural L-amino acids in the structure of CPPs. The substitution made CPP be more resistant against degradation by environmental enzymes.2 Doxorubicin (Dox) has been extensively utilized in the treatment of various tumors and was designated as a model antitumor agent because of its excellent efficacy by suppressing the topoisomerase II and inducing cell apoptosis. However, its clinical application is complicated by its severe off-target toxicity and acquired drug resistance. CPP-Dox conjugates through coupling CPP with Dox have been extensively applied and have demonstrated the effectiveness for the therapy of various tumors.28−30 CPP-Dox and mCPP-Dox were synthesized by Michael addition reaction using a linker of SMP. The synthetic route was described in Figure 2. SMP-Dox derivative was prepared initially based on the principle of active ester reaction according to previous method.22 Cysteine in C-terminal amino acid of the CPP or mCPP is a chemically reactive group and is easy to conjugate with drug. On the basis of the principle of Michael addition (nucleophilic addition) reaction, CPP-Dox or mCPPDox can be prepared by covalently coupling maleimide of SMPDox derivative with the cysteine sulfur in peptides. SMP-Dox derivative: MALDI-TOF-MS (m/z, [M + Na]+): 717.6 (theoretical), 715.8 (practical); MALDI-TOF-MS (m/z, [M E
DOI: 10.1021/acsami.7b01056 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
Figure 4. Tumor volume change (1), body weight change (2), and tumor weight (3) in the therapeutic schedule I (A, n = 7), II (B, n = 9−10), and III (C, n = 6). Treatment groups included physiological saline, mCPP-Dox, CPP-Dox, and free Dox. Animal survival percentage change was shown in internal figure of C1 during the therapy of schedule III. Error bars show the standard deviation. *p < 0.05; **p < 0.01; ***p < 0.001. n.s. = no significant difference.
mainly focuses on a minimum value at 218 nm and a maximum value at 198 nm.31 As shown in Figure 3A, mCPP and mCPPDox showed a trough at 216.6 and 215.2 nm, respectively, which indicated the presence of beta-sheet structure and there was no specific tertiary structure. On the basis of the analysis of CD data, before reaching to the target site and being triggered by MMP-2/9, mCPP-Dox tends to adopt beta-sheet secondary structure and is favorable to be triggered by MMP-2/9 in tumor tissues. This result was consistent with the spatial structure of the mCPP building by Biopolymer module in Insight II (Figure S7, SI). 3.3. Exogenous MMP-2/9 triggers the reversion of masked CPP-cargo to CPP-cargo in accordance with Michaelis−Menten kinetic profile. Masked CPP-cargo can curb the wildness of CPP-cargo until encountering MMP-2/9 in tumor tissue. For mCPP-Dox conjugate, protease (MMP-2/ 9) is a trigger to unlock this conjugate. The reversion efficiency of CPP-cargo in conjugates depends on the enzymatic degradation kinetics. Exogenous MMP-2/9 was added into mCPP-Dox conjugate solution to explore whether the conjugate could be cleaved by MMP-2/9 and how the
conjugate was cleaved by MMP-2/9. When MMP-2/9 is employed, mCPP-Dox could be cleaved and the half-time of cleavage decreased as the MMP-2/9 concentration increased (Figure 3B). The change of the triggered cleavage half-time (y) and the ratio (x) followed the Equation 1: y = −1.2151 ln(x) − 0.5247
(1)
Actually, most enzyme-guided catalysis reactions also follow Michaelis−Menten kinetics model.32−34 The degradation reaction between enzyme (MMP-2/9) and substrate (mCPPDox conjugate) was assumed to be of the common Michaelis− Menten type (eq 2): k1
S + E ⇌ ES ⇀ P + E k2
(2)
where initially, the enzyme (E) attached to the conjugate substrate (S) and formed an enzyme−substrate complex (ES). Enzyme scissors substrate, the product (P) and enzyme itself were released. The degraded products of mCPP-Dox were CPP-Dox and shielding peptide sequences. The degradation kinetic equation was eq 3: F
DOI: 10.1021/acsami.7b01056 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
Figure 5. Hematological indicators were measured including WBC (1), HGB (2), RBC (3), and PLT (4) at Day 21 (A) in the therapeutic schedule II, Day 10 (B) and Day 19 (C) in the therapeutic schedule III. Error bars indicate the standard deviation.
VmC dC =− dt (K m + C )
concentration range, mCPP or CPP did not produce an obvious toxicity against both two cell lines (data not shown). With the increase of the drug concentration, the antiproliferative activities of free Dox increased significantly. For Dox, the IC50 values in HT-1080 and MCF-7 cells were 21.6 μM and 18.6 μM at 24 h, 1.5 μM and 1.3 μM at 48 h, respectively (Figure 3D). The sensitiveness of various compounds toward endogenous MMP-2/9 could be evaluated by IC50 ratio in HT1080 and MCF-7 cells (see the inset in Figure 3D). A ratio value of approximately 1 indicates that the parent drug has similar antiproliferative activity against both cell lines. The IC50 ratio of Dox in HT-1080 and MCF-7 cells was 1.16 at 24 h and 1.15 at 48 h, respectively, that is , Dox has no preference for both types of cells. Similarly, the IC50 ratio of CPP-Dox conjugate was 1.18 at 24 h. The indiscriminate cytotoxicity of the CPP-Dox conjugates at MMPs-rich HT-1080 cells and MMPs-depleted MCF-7 cells showed their wildness or nonselectivity toward different MMP-2/9-secreted cell lines. However, the IC50 ratio of mCPP-Dox was 0.59 in HT-1080 at 24 h, and 0.50 in MCF-7 at 48 h. Apart from Dox and CPPDox conjugate, the lower ratio (
