Article Redox-Sensitive Nanocomplex for Targeted Delivery of

Bei Cheng and Peisheng Xu *

Department of Discovery and Biomedical Sciences, College of Pharmacy, University of South Carolina, 715 Sumter, Columbia, SC 29208, USA; [email protected] * Correspondence: [email protected]

 Received: 10 August 2020; Accepted: 8 September 2020; Published: 10 September 2020 

Abstract: Although therapeutics have been explored for decades, the successful delivery of potent in vitro and in vivo remains challenging due to the poor stability, low cell permeability, and off-target effects. We developed a redox sensitive polymer-based nanocomplex which can efficiently and stably deliver the peptide drug melittin for therapy. The nanocomplex selectively targets cancer cells through lactobionic acid mediated endocytosis and releases melittin intracellularly upon the trigger of elevated redox potential. In vivo study proved that the targeted nanocomplex shows excellent potency in inhibiting tumor growth in a xenograft colon cancer mouse model. Thus, the polymer/melittin nanocomplexes will provide a new approach for melittin based cancer therapy.

Keywords: nanocomplex; targeted delivery; melittin; redox sensitive; cancer

Key Contribution: A polymer/melittin nanocomplex was developed to selectively deliver melittin into cancer cells and release intracellularly due to the endogenous high redox potential. This redox sensitive polymer/melittin nanocomplex will provide a new approach for melittin based cancer therapy.

1. Introduction Peptide-based drugs, which usually have less than 50 amino acids, hold a high potential for treating various diseases. Furthermore, some peptide-based drugs have successfully entered the market, such as cyclosporine, which is routinely used after organ transplantation to reduce the activity of the immune system [1]. Some peptides can also be utilized as a cytosolic agent. One of the most well-established peptides is luteinizing hormone-releasing hormone agonists for the combination treatment of prostate cancer combined with radiation or chemotherapy [2,3]. The last decade has witnessed the exponential growth of many peptide drugs entering clinical trials [4]. Specifically, peptides hold many advantages for anticancer due to their high potency and a broad range of targets. However, many hurdles, including vulnerability toward proteolytic digestion and side effects due to off-targeting, have yet to be overcome [5–7]. For instance, the available cyclosporine preparations showed a low bioavailability, high variation between patients, and the inability to obtain both effective and safe doses [1]. Recently, melittin, a natural peptide from composed of 26 amino acids, has attracted a lot of attention due to its anti-arthritis, anti-inflammatory, anticancer, and neuroprotective properties [8–13]. Melittin self-assembles into toroidal structures and induces the formation of pores on the cell membrane, which ultimately results in cell death [14,15]. This unique cytolytic feature makes it impossible for cancer cells to develop drug resistance. Due to the same reason, the non-specific cytolytic activity of melittin could result in severe off-target effects, such as hemolysis (lysis of red blood cells), when administrated intravenously. To make melittin druggable and safer, many approaches have been explored through

Toxins 2020, 12, 582; doi:10.3390/toxins12090582 www.mdpi.com/journal/toxins Toxins 2020, 12, x FOR PEER REVIEW 2 of 14 ofToxins red2020 blood, 12, 582cells), when administrated intravenously. To make melittin druggable and safer, many2 of 13 approaches have been explored through environmental liable modification [16,17] or nanoencapsulation [18–20]. Previously, our group utilized a dual-secured “nanobee” technology withenvironmental the help liableof succinic modification anhydride-modified [16,17] or nanoencapsulation glycol chitosan [18 –20(SAMGC),]. Previously, and ourdisulfide group utilizedbonds successfullya dual-secured increased “nanobee” the technologytherapeutic withwindow thehelp of melittin of succinic to 10 anhydride-modified µM [21]. The positively glycol chitosancharged melittin(SAMGC), forms and disulfidenanocomplexes bonds successfullywith a negatively increased charged the therapeutic SAMGC windowthrough ofthe melittin electrostatic to 10 µ M[effect,21]. whileThe positively the disulfide charged bonds melittin ensure forms that nanocomplexes the encapsulated with melittin a negatively can chargedonly be SAMGCreleased throughinside the the cytosol,electrostatic where eff ect,it has while elevated the disulfide glutathione bonds (GSH ensure), to that prevent the encapsulated the occurrence melittin of hemolysis. can only be released insideOur the cytosol,group whererecently it hasdeveloped elevated glutathionea poly[(2-(py (GSH),ridin-2-yldisulfanyl) to prevent the occurrence ethyl ofacrylate)-co- hemolysis. [poly(ethyleneOur group glycol)]] recently (PDAPEG) developed apolymer poly[(2-(pyridin-2-yldisulfanyl) [22–24], which can be easily ethyl modified acrylate)-co-[poly(ethylene by thiol-disulfide exchangeglycol)]] (PDAPEG) reaction. Thanks polymer to [ 22this–24 nature,], which a canserial be of easily zwitterionic modified polymers by thiol-disulfide with different exchange isoelectric reaction. pointsThanks (IEPs) to this could nature, be produced a serial of by zwitterionic simply tuning polymers the ratio with of di thefferent introduced isoelectric NH points2 and COOH (IEPs) couldgroups. be Sinceproduced the intracellular by simply tuning environment the ratio usually of the introduced contains a NH1000-fold2 and COOH higher groups.level of SinceGSH than the intracellular that in the extracellularenvironment milieus usually [25], contains the high a 1000-fold redox potential higher level can of be GSH utilized than to that trigger in the th extracellulare release of peptides milieus [ 25by], breakingthe high redoxthe disulfide potential bonds. can be utilized to trigger the release of peptides by breaking the disulfide bonds. ToTo improve improve the the specificity specificity for for cancer cancer targeted targeted delivery, delivery, various various cancer cancer cell-related cell-related receptors receptors have beenhave explored. been explored. Among Amongthem, a β them,-D-galactose a β-D-galactose receptor, also receptor, known also as ASGPR1, known as has ASGPR1, been extensively has been investigated.extensively investigated. It was revealed It that was ASGPR1 revealed receptor that ASGPR1 is expressed receptor in multiple is expressed typesin of multiplecancer, including types of ovariancancer, includingcancer, liver ovarian cancer, cancer, breast liver cancer, cancer, and breastcolon cancer,cancer [26–28]. and colon Theref cancerore, [ 26many–28]. ligands Therefore, for ASGRP1many ligands receptor for ASGRP1have been receptor developed, have including been developed, lactobionic including acid (LBA), lactobionic for cancer acid targeted (LBA), for therapy cancer [29].targeted Colon therapy cancer [ can29]. develop Colon cancer acquired can multidrug develop acquired resistance multidrug during conventional resistance during treatment conventional [30] and hastreatment been validated [30] and hasfor LBA been targeting validated in for our LBA previous targeting research. in our previous research. Herein,Herein, we we developed developed a a redox redox responsive, responsive, ASGR ASGRPP targeted targeted polymer/melittin polymer/melittin nanocomplex, nanocomplex, for for thethe treatment treatment of of colon colon cancer. cancer. The The negatively negatively charged PDAPEG polymer derivative derivative forms forms complex complex melittinmelittin through through electrostatic force. The The nanoco nanocomplexesmplexes enter enter cancer cancer cells cells through through LBA LBA mediated endocytosis,endocytosis, subsequently subsequently dissociate dissociate due due to to the the cl cleavageeavage of of disulfide disulfide bonds, bonds, and and release release melittin intracellularlyintracellularly to to kill kill cancer cancer cells cells (Figure (Figure 11).). Since there is no prematurepremature releaserelease ofof melittinmelittin beforebefore itit entersenters cancer cancer cells, cells, we we believe believe this this nanocomplex nanocomplex ca cann translate translate targeted targeted melittin melittin therapy therapy to to a a clinically clinically promisingpromising treatment with high efficiency efficiency and low side eeffects.ffects.

Figure 1. Delivery pathway of targeted melittin nanocomplex for cancer therapy.

2. Results and Discussion Figure 1. Delivery pathway of targeted melittin nanocomplex for cancer therapy. 2.1. Design and Synthesis of Polymer LBA-PDAPEG

In our recent work, we prepared an environment-sensitive peptide delivery system, dual secured nano-sting through the combination of a zwitterionic glycol chitosan and disulfide bond [21]. In that design, melittin was successfully delivered into cytosol and released under the acidic pH and high Toxins 2020, 12, x FOR PEER REVIEW 3 of 14

2. Results and Discussion

2.1. Design and Synthesis of Polymer LBA-PDAPEG

Toxins In2020 our, 12, 582recent work, we prepared an environment-sensitive peptide delivery system, 3dual of 13 secured nano-sting through the combination of a zwitterionic glycol chitosan and disulfide bond [21]. In that design, melittin was successfully delivered into cytosol and released under the acidic pH and redoxhigh redox environment. environment. In this In study, this study, we aimed we toaimed deliver to melittindeliver melittin through through a redox sensitivea redox polymersensitive PDAPEG,polymer PDAPEG, as shown as in shown Figure 1in. Figure 1. TheThe synthesis ofof LBA-PDAPEG isis described inin Figure2 2.. ToTo introduceintroduce carboxylic carboxylic acid acid groups groups and and amineamine groupsgroups intointo thethe PDA-PEG polymer, thethe pyridinepyridine ringring waswas replacedreplaced completelycompletely byby cysteaminecysteamine oror 3-mercaptopropionic3-mercaptopropionic acidacid throughthrough thiolthiol disulfidedisulfide exchangeexchange reaction.reaction. The complete of the reaction waswas confirmedconfirmed byby UV–visUV–vis andand 11H-NMR.H-NMR. The signaturesignature peakpeak ofof 2-pyridinethiol2-pyridinethiol at 375 nmnm afterafter cleavagecleavage byby tris(2-carboxyethyl)phosphine (TCEP)(TCEP) inin thethe UV–vis spectrumspectrum disappeareddisappeared afterafter thethe thiol disulfidedisulfide exchangeexchange inin FigureFigure3 3A.A. The The disappeared disappeared protons protons in in pyridine pyridine rings rings (8.46, (8.46, 7.69, 7.69, 7.11 7.11 ppm) ppm) in in thethe 1H-NMR spectrum, spectrum, as as shown shown in in Figure Figure 3B,3B, support the the conclusion. conclusion. Successful Successful conjugation conjugation of of 3- 3-mercaptopropionicmercaptopropionic acid acid was was characterized characterized by by the the –CH –CH2CH2CH2–2 protons– protons (2.95, (2.95, 2.74 2.74 ppm). ppm).

FigureFigure 2.2. The synthesis scheme of LBA-PDAPEG.

The composition of amine to carboxylic acid group determines the isoelectric point (IEP) of polymer, which can be tuned by varying the feeding ratio of cysteamine to 3-mercaptopropionic acid. The IEP points of polymers ranged from 9.5 to 5.0 when the cysteamine to 3-mercaptopropionic acid was fed from 4/1 to 1/4 (Figure4). The wide range of tunable surface charge of the polymers would allow for the binding of peptides with different IEPs. The gentle electrostatic force between polymer and peptide would prevent the potential denature of peptide if prepared with the traditional encapsulation method accompanied with high intensive mechanical force or heat. In our study, we used a PDAPEG derivative with a surface charge around 11 mV at the pH of 7.4 for nanocomplex fabrication. The targeting − moiety of LBA was conjugated to the amine group of through N-hydroxysuccinimide (NHS) and Toxins 2020, 12, 582 4 of 13

1-thyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC) reaction. The conjugation was quantitatively determined by the reduction of amine amount through a 2,4,6-trinitrobenzene sulfonic acid (TNBSA) assay. The unmodified PDAPEG was determined to have 500 nmol pyridine rings per 1 mg polymer with the co-incubation with 10 mM dithiothreitol (DTT). After the modification of both by cysteamine and mercaptopropionic acid, the total amine amount was increased to 40.6 nmol per mg polymer. ToxinsAfter 2020 the,conjugation 12, x FOR PEER of REVIEW LBA, the available amine was decreased to 32.5 nmol per mg polymer, which4 of 14 indicates a successful conjugation of LBA.

Figure 3. ((A).). UV–Vis spectrumspectrum of polymerpolymer PDAPEGPDAPEG or PDAPEG-COOHPDAPEG-COOH when co-incubating with 10 mM TCEP. The appeared peak at 375 nm indicatesindicates the existence of of pyridinethiol pyridinethiol in in polymer; polymer; ( (BB).). 1H-NMR of polymer PDAPEG-COOH, PDAPEG-NH , and PDAPEG in CDCl . 1H-NMR of polymer PDAPEG-COOH, PDAPEG-NH22, and PDAPEG in CDCl33. 2.2. Particle Characterization The composition of amine to carboxylic acid group determines the isoelectric point (IEP) of polymer,Through which electrostatic can be tuned complexation, by varying the a feeding negatively ratiocharged of cysteamine PDAPEG to 3-mercaptopropionic derivative formed acid. with Thecomplexed IEP points positively of polymers charged ranged peptide from melittin 9.5 to 5.0 at physiologywhen the cysteamine pH and further to 3-mercaptopropionic condensed it. After acid the wasformation fed from of complex, 4/1 to 1/4 melittin (Figure was 4). The entrapped wide range within of the tunable network surface of nano-structure charge of the and polymers was protected would allowthrough for thethe outlayerbinding of hydration peptides of with PEG. different The nanocomplex IEPs. The gentle showed electrostatic a particle force hydrodynamic between polymer size of 357 30 nm (Figure5). They were almost spherical according to transmittance electron microscopy. and ±peptide would prevent the potential denature of peptide if prepared with the traditional In addition, the surface charge of nanocomplex dropped from 11.8 0.64 to 4.24 0.21 mV also encapsulation method accompanied with high intensive mechanical− force± or heat.− In± our study, we usedindicates a PDAPEG the successful derivative complexation with a surface with charge melittin. around This -11 proper mV at size the would pH of allow7.4 for for nanocomplex the passive fabrication.transportation The of targeting nanoparticles moiety entering of LBA cancer was cells conjugated through enhancedto the amine permeability group of and through retention N- hydroxysuccinimideeffect [31]. (NHS) and 1-thyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC) reaction. The conjugation was quantitatively determined by the reduction of amine amount through a 2,4,6- trinitrobenzene sulfonic acid (TNBSA) assay. The unmodified PDAPEG was determined to have 500 nmol pyridine rings per 1 mg polymer with the co-incubation with 10 mM dithiothreitol (DTT). After the modification of both by cysteamine and mercaptopropionic acid, the total amine amount was increased to 40.6 nmol per mg polymer. After the conjugation of LBA, the available amine was decreased to 32.5 nmol per mg polymer, which indicates a successful conjugation of LBA.

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PDAPEG (NH2:COOH-4:1) PDAPEG (NH2:COOH-1:4) PDAPEG (NH2:COOH-1:1) PDAPEG (NH2:COOH=2:1) 30 PDAPEG (NH2:COOH=1:2) PDA-co-PEG-COOH PDAPEG (No modification) PDAPEG-NH2 20

10

0 2.54.56.58.510.5 Toxins 2020, 12, 582 5 of 13 δ-potential (mV) δ-potential Toxins 2020, 12, x -10FOR PEER REVIEW pH 5 of 14

-20 PDAPEG (NH2:COOH-4:1) PDAPEG (NH2:COOH-1:4) PDAPEG (NH2:COOH-1:1) PDAPEG (NH2:COOH=2:1) 30 PDAPEG (NH2:COOH=1:2) PDA-co-PEG-COOH -30 PDAPEG (No modification) PDAPEG-NH2 20 Figure 4. The surface charges of PDAPEG-NH2/PDAPEG-COOH at different amine to carboxylate ratio responsive to environmental pH. 10 2.2. Particle Characterization 0 Through electrostatic2.54.56.58.510.5 complexation, a negatively charged PDAPEG derivative formed with

complexed positively(mV) δ-potential charged peptide melittin at physiology pH and further condensed it. After the -10 pH formation of complex, melittin was entrapped within the network of nano-structure and was protected through the outlayer hydration of PEG. The nanocomplex showed a particle hydrodynamic size of 357 ± 30-20 nm (Figure 5). They were almost spherical according to transmittance electron microscopy. In addition, the surface charge of nanocomplex dropped from −11.8 ± 0.64 to −4.24 ± 0.21 -30 mV also indicates the successful complexation with melittin. This proper size would allow for the passive transportation of nanoparticles entering cancer cells through enhanced permeability and FigureFigure 4. 4. TheThe surface surface charges charges of of PDAPEG-NH2/PDAPEG- PDAPEG-NH2/PDAPEG-COOHCOOH at at different different amine amine to to carboxylate carboxylate retentionratioratio effect responsive responsive [31]. to to environmental environmental pH. pH.

2.2. Particle CharacterizationA B Through electrostatic complexation, a negatively charged PDAPEG derivative formed with 2020 complexed positively charged peptide melittin at physiology pH and further condensed it. After the formation of complex, melittin was entrapped within the network of nano-structure and was 1515 protected through the outlayer hydration of PEG. The nanocomplex showed a particle hydrodynamic size of 357 ± 30 nm (Figure 5). They were almost spherical according to transmittance electron 10 microscopy. In addition,10 the surface charge of nanocomplex dropped from −11.8 ± 0.64 to −4.24 ± 0.21

mV also indicatesIntensity the successful complexation with melittin. This proper size would allow for the 55 passive transportation of nanoparticles entering cancer cells through enhanced permeability and retention effect [31]. 00 10 100 1000 A Size (d. nm) B

Figure 5. Characterization2020 of the nanocomplexes. Hydrodynamic size of nanocomplex in TBS buffer Figuremeasured 5. Characterization by dynamic light of the scattering nanocomplexes. (A) and TEMHydrod imageynamic of nanocomplex size of nanocomplex (B). Scale in barTBS inbuffer (B) is measured by dynamic light scattering (A) and TEM image of nanocomplex (B). Scale bar in (B) is 200 200 nm. 1515 nm. 2.3. High Redox Potential Environment Could Trigger the Release of Melittin 1010 2.3. HighA unique Redox Potential feature ofEnvironment intercellular Could environment Trigger the is Release its high of reduction Melittin potential due to the elevated

GSH, which playsIntensity an important role in maintaining the reducing environment to prevent the excessive A unique feature55 of intercellular environment is its high reduction potential due to the elevated GSH,oxidation which stress plays from an normal important activities role suchin maintaining as electron transfer the reducing from respiration environment or photosynthesis to prevent the [32 ]. Our polymer LBA-PDAPEG would be almost neutral after exposing it to a high reduction environment excessive oxidation 00stress from normal activities such as electron transfer from respiration or due to the lost functionalization of carboxylate group and amine group. Thus, the complex would be 10 100 1000 dissociated quickly and would releaseSize (d. melittin nm) accordingly. To evaluate how the complex responds to a high GSH environment, fluorescence resonance energy transfer (FRET) based nanocomplex was

prepared as described, using Cy3 as energy donor and Cy5 as energy receptor. Excited at 530 nm of Cy3,Figure the energy 5. Characterization was transferred of the from nanocomplexes. PDAPEG-Cy3 Hydrod (donor)ynamic to melittin-Cy5size of nanocomplex (acceptor), in TBS which buffer led to measured by dynamic light scattering (A) and TEM image of nanocomplex (B). Scale bar in (B) is 200 an increased emission intensity at 670 nm. A reducing environment, such as 10 mM DTT, was able nm. to cleave carboxylate groups. The dissociation of neutral polymer and positive melittin led to a decreased energy transfer, which decreased the fluorescence intensity at 660 nm as shown in Figure6. 2.3. High Redox Potential Environment Could Trigger the Release of Melittin The fluorescence intensity was reduced by 20% after 50 min incubation with DTT, suggesting that the nanocomplexA unique integrityfeature of was intercellular partially compromisedenvironment is by its a high reducingreductionenvironment. potential due to the elevated GSH, which plays an important role in maintaining the reducing environment to prevent the excessive oxidation stress from normal activities such as electron transfer from respiration or

Toxins 2020, 12, x FOR PEER REVIEW 6 of 14 photosynthesis [32]. Our polymer LBA-PDAPEG would be almost neutral after exposing it to a high reduction environment due to the lost functionalization of carboxylate group and amine group. Thus, the complex would be dissociated quickly and would release melittin accordingly. To evaluate how the complex responds to a high GSH environment, fluorescence resonance energy transfer (FRET) based nanocomplex was prepared as described, using Cy3 as energy donor and Cy5 as energy receptor. Excited at 530 nm of Cy3, the energy was transferred from PDAPEG-Cy3 (donor) to melittin- Cy5 (acceptor), which led to an increased emission intensity at 670 nm. A reducing environment, such as 10 mM DTT, was able to cleave carboxylate groups. The dissociation of neutral polymer and positive melittin led to a decreased energy transfer, which decreased the fluorescence intensity at 660 nm as shown in Figure 6. The fluorescence intensity was reduced by 20% after 50 min incubation with DTT, suggesting that the nanocomplex integrity was partially compromised by a high reducing Toxinsenvironment.2020, 12, 582 6 of 13

AB 1.6

1.4

0.6

F660/F560*10 0.4 0.2 0.0 10 20 30 40 50 60 Time (min)

Figure 6. The measured FRET intensities of nanocomplex with or without DTT as a function of time Figure 6. The measured FRET intensities of nanocomplex with or without DTT as a function of time (A) and full spectrum of complex with or without DTT when measured the FRET intensity (B). (A) and full spectrum of complex with or without DTT when measured the FRET intensity (B). 2.4. Hemolytic Assay 2.4. Hemolytic Assay To evaluate the safety of the polymer/melittin nanocomplex, hemolysis assay was adopted. Free melittinTo evaluate was the able safety to lysis of morethe polymer/melittin than 80% of red nanocomplex, blood cells (RBCs) hemolysis at the concentrationassay was adopted. of 0.5 FreeµM, whereasmelittin thewas hemolytic able to lysis activity more of than the polymer80% of red/melittin blood nanocomplexcells (RBCs) at decreased the concentration with the increaseof 0.5 µM, of polymerwhereas tothe melittin hemolytic ratio activity (Figure of7 theA). polymer/meli No RBC lysisttin was nanocomplex observed for decreased the nanocomplexes with the increase formed of atpolymer the polymer to melittin/melittin ratio ratio (Figure of 307A). at No the RBC melittin lysis concentration was observed of for 5 theµM, nanocomplexes indicating the formed excellent at the polymer/melittin ratio of 30 at the melittin concentration of 5 µM, indicating the excellent safety safety of the nanocomplexes. As expected, after the co-incubation with 10 mM GSH at 37 ◦C for 2 h, theof the nanocomplex nanocomplexes. solution As lysedexpected, 40% andafter 100% the co-incubation of RBCs at 2 µwithM and 10 5mMµM, GSH respectively at 37 °C (Figurefor 2 h,7 B).the Itnanocomplex is believed that solution the complex lysed 40% was and dissociated 100% of upon RBCs the at 2 increased µM and reducing5 µM, respectively environment (Figure and released7B). It is believed that the complex was dissociated upon the increased reducing environment and released theToxins free 2020 melittin, 12, x FOR to PEER form REVIEW pores on the surface of RBCs and lysis them. 7 of 14 the free melittin to form pores on the surface of RBCs and lysis them.

Figure 7. Hemolytic activity of melittin and nanocomplex. (A) The hemolytic activity of Figure 7. Hemolytic activity of melittin and nanocomplex. (A) The hemolytic activity of PDAPEG/melittin nanocomplexes of different polymer to melittin ratios. (B) The hemolytic activity of PDAPEG/melittin nanocomplexes of different polymer to melittin ratios. (B) The hemolytic activity melittin, PDA-PEG, and PDAPEG/melittin nanocomplexes in different pH and reducing environments. of melittin, PDA-PEG, and PDAPEG/melittin nanocomplexes in different pH and reducing *** p < 0.001, **** p < 0.0001. environments. *** p < 0.001, **** p < 0.0001. 2.5. In vitro Cellular Uptake 2.5. In vitro Cellular Uptake It has been reported that LBA can bind to cancer cells membrane overexpressed D-galactose receptors.It has To been investigate reported the that cellular LBA uptakecan bind of LBA-nanocomplexes,to cancer cells membrane confocal overexpressed microcopy wasD-galactose applied. Withreceptors. a short To term investigate of incubation the with cellular HCT 116uptake cells, LBA-modifiedof LBA-nanocomp nanocomplexeslexes, confocal showed microcopy a significantly was applied. With a short term of incubation with HCT 116 cells, LBA-modified nanocomplexes showed a significantly increased uptake compared with the non-targeted group, as evidenced by the brighter red fluorescent (Figure 8). This data indicated that LBA could serve as a targeting moiety toward HCT 116 colon cells. A similar LBA based targeting effect was also observed in the MCF-7 cells (data not shown).

Toxins 2020, 12, 582 7 of 13

increased uptake compared with the non-targeted group, as evidenced by the brighter red fluorescent (Figure8). This data indicated that LBA could serve as a targeting moiety toward HCT 116 colon cells. ToxinsA 2020 similar, 12, x FOR LBA PEER based REVIEW targeting effect was also observed in the MCF-7 cells (data not shown).8 of 14

Figure 8. Confocal microscope images of HCT-116 colon cancer cells after incubating with control Figurenanocomplex 8. Confocal ormicroscope LBA-nanocomplex images of for HCT-116 3 h. colon cancer cells after incubating with control nanocomplex or LBA-nanocomplex for 3 h.

2.6. In vitro Anticancer Activities The of nanocomplex was investigated toward various cancer cells, including HCT 116 and MCF-7 cells, as shown in Figure 9. Compared with free melittin, non-targeted nanocomplex showed a similar effect at low concentration but less effective at concentration higher than 1 µM in

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2.6. In vitro Anticancer Activities

ToxinsThe 2020 cytotoxicity, 12, x FOR PEER of REVIEW nanocomplex was investigated toward various cancer cells, including9 HCT of 14 116 and MCF-7 cells, as shown in Figure9. Compared with free melittin, non-targeted nanocomplex showedkilling cancer a similar cells. eff Remarkably,ect at low concentration the LBA-nanocomp but lesslex eff ectiveshowed at an concentration enhanced cytotoxicity higher than compared 1 µM in killingto non-targeted cancer cells. nanocomplex Remarkably, theand LBA-nanocomplex also significantly showed higher an potency enhanced than cytotoxicity free melittin compared at all to non-targetedconcentrations. nanocomplex For HCT 116 and cells, also the significantly IC 50 for nanocomplex higher potency was than 4.15 free µM melittin and the at LBA-nanocomplex all concentrations. Forwas HCT1 µM. 116 The cells, targeting the IC effect 50 for contributed nanocomplex a 76% was decrease. 4.15 µM Although and the LBA-nanocomplex melittin is more toxic was toward 1 µM. TheMCF-7 targeting cells, ewhichffect contributed killed slightly a 76% less decrease. than 90% Although of cells melittin at the is moreconcentration toxic toward of 5 MCF-7 µM, LBA- cells, whichnanocomplex killed slightly could lessalmost than eradicate 90% of cells all atcancer the concentrationcells at the ofsame 5 µ M,dose. LBA-nanocomplex Most melittin-loaded could almostnanoparticles eradicate exhibit all cancer similar cells or at lower the same potency dose. than Most free melittin-loaded melittin in killing nanoparticles cancer cells exhibit [21,33]. similar Based or loweron our potency findings than and free the melittin reported in killing results cancer from cells other [21 ,33research]. Based groups, on our findingswe conclude and the that reported LBA- resultsnanocomplex from other exhibited research a better groups, efficacy we concludetoward vari thatous LBA-nanocomplex cancer cells than free exhibited melittin a bettermainly e ffiduecacy to towardits cancer-targeting various cancer effect cells [34]. than free melittin mainly due to its cancer-targeting effect [34].

A B

C

Figure 9. Cytotoxicity of melittin, control nanocomplex, and LBA-nanocomplex for various cancer Figure 9. Cytotoxicity of melittin, control nanocomplex, and LBA-nanocomplex for various cancer cells. Cells were incubated with melittin nanocomplex and LBA-nanocomplex at melittin concentration cells. Cells were incubated with melittin nanocomplex and LBA-nanocomplex at melittin from 0.1 to 10 µM for 24 h. Data represent mean SD, (A) HCT 116 cell; (B) MCF-7 cells; (C) Polymer concentration from 0.1 to 10 µM for 24 h. Data represent± mean ± SD, (A) HCT 116 cell; (B) MCF-7 cells; cytotoxicity for HCT-116 and MCT-7 cells. * p < 0.05, ** p < 0.01, *** p < 0.001. (C) Polymer cytotoxicity for HCT-116 and MCT-7 cells. * p < 0.05, ** p< 0.01, *** p < 0.001. 2.7. In Vivo Experiment 2.7. In Vivo Experiment To assess the tumor growth inhibitory effect of the nanocomplex, the in vivo experiment was carriedTo outassess in a the xenograft tumor HCTgrowth 116 inhi colonbitory tumor-bearing effect of the nude nanocomplex, mouse model. the Figurein vivo 10 experimentA revealed thatwas polymercarried out/melittin in a xenograft nanocomplexes HCT 116 remained colon tumor-bearing active. Both nude the non-targeted mouse model. and Figure targeted 10A nanocomplex revealed that canpolymer/melittin effectively inhibit nanocomplexes the growth ofrema tumors.ined active. After twoBoth weeks the non-targeted of treatment, and the targeted size of tumorsnanocomplex in the nanocomplexescan effectively inhibit treated the groups growth was of tumors. around halfAfter of two the weeks tumor of size treatment, in the control the size group. of tumors After in thethe sacrificenanocomplexes of the animals, treated the groups tumors was were around isolated half and of weighed.the tumor Figure size in10 Bthe indicated control thegroup. tumor After weight the sacrifice of the animals, the tumors were isolated1 and weighed. Figure 10B indicated the tumor of LBA nanocomplex treated group was less than 4 of that in the control group. Consequently, the LBA nanocomplexweight of LBA showed nanocomplex a significantly treated better group tumor was growth less inhibitorythan ¼ eofff ectthat than in thethe control. control group. Consequently, the LBA nanocomplex showed a significantly better tumor growth inhibitory effect than the control.

Toxins 2020, 12, 582 9 of 13 Toxins 2020, 12, x FOR PEER REVIEW 10 of 14

Figure 10. Tumor growth inhibitory effect of the LBA nanocomplex. Tumor growth profile (A) and Figure 10. Tumor growth inhibitory effect of the LBA nanocomplex. Tumor growth profile (A) and tumor weight (B) after receiving different treatments. Representative tumor images of each group at tumor weight (B) after receiving different treatments. Representative tumor images of each group at the end of treatment (insert). * p < 0.05. the end of treatment (insert). * p < 0.05. 3. Conclusions 3. Conclusions In this study, we successfully developed a polymer/melittin based nanocomplex to evaluate its safetyIn andthis estudy,fficacy we in successfully cancer therapy. developed It was revealeda polymer/melittin that the nanocomplex based nanocomplex can effectively to evaluate deliver its safetymelittin and into efficacy cancer cellsin cancer and releasetherapy. its contentsIt was revealed responding that tothe high nanocomplex redox potential can environmenteffectively deliver while melittinprotecting into RBCs cancer from cells the and damage release of freeits contents melittin. responding The LBA-nanocomplex to high redox is morepotential effective environment in killing whileboth HCT protecting 116 colon RBCs cancer from cells the damage and MCF-7 of free breast melittin. cancer The cells. LBA-nanocomplex Furthermore, the isin more vivo experimenteffective in killingproved both that theHCT LBA-nanocomplex 116 colon cancer can cells effectively and MCF-7 kill cancer breast cells, cancer thus resultingcells. Furthermore, in significantly the reducedin vivo experimenttumor progression proved inthat a xenograft the LBA-nanocomplex colon cancer mouse can e model.ffectively This kill redox cancer sensitive cells, thus polymer resulting/melittin in significantlynanocomplexes reduced will provide tumor aprogression new approach in fora xenograft melittin based colon cancer cancer therapy. mouse model. This redox sensitive polymer/melittin nanocomplexes will provide a new approach for melittin based cancer therapy.4. Materials and Methods

4.4.1. Materials Materials and Methods Melittin was purchased from SERVA Electrophoresis GmbH (Heidelberg, Germany); 4.1.3-mercaptopropionic Materials acid, aldrithiol-2, acryloyl chloride, poly(ethylene glycol)methacrylate (Mn Melittin= 360 Da ),was cysteamine purchased hydrochloride, from SERVA hydrochloride Electrophoresis acid, GmbH trimethylamine, (Heidelberg, 2-(N-morpholino) Germany); 3- mercaptopropionicethanesulfonic acid acid, (MES), aldrithiol-2, 2,2-azobis(2-methylpropionitrile) acryloyl chloride, poly(ethylene (AIBN), glycol)methacrylate lactobionic acid (Mn (LBA), = 360 Da),N-hydroxysuccinimide cysteamine hydrochloride, (NHS), and cysteaminehydrochloride hydrochloride acid, trimethylamine, were purchased from2-(N-morpholino) Sigma-Aldrich ethanesulfonic(St. Louis, MO, acid USA). (MES), EDC (1-thyl-3-(3-(dimethylamino)propyl)2,2-azobis(2-methylpropionitrile) (AIBN), carbodiimide) lactobionic was purchasedacid (LBA), from N- hydroxysuccinimideTCI (TCI America, Domorah, (NHS), and PA, cy USA);steamine 2,4,6-trinitrobenzene hydrochloride were sulfonic purchased acid was from purchased Sigma-Aldrich form Life(St. Louis,Technologies. MO, USA). Sodium EDC dodecyl (1-thyl-3-(3-(dimethylamin sulfate (SDS) was purchasedo)propyl) from carbodiimide) Bio-Rad Laboratories, was purchased Inc (Hercules,from TCI (TCICA). America, HCT 116 Domorah, human colorectal PA, USA); cancer 2,4,6-trinitro cells andbenzene MCF-7 sulfonic breast cancer acid was cells purchased were obtained form fromLife Technologies.American Type Sodium Culture Collectiondodecyl sulfate (ATCC). (SDS) was purchased from Bio-Rad Laboratories, Inc (Hercules, CA). HCT 116 human colorectal cancer cells and MCF-7 breast cancer cells were obtained 4.2. Preparation of Melittin Polymer from American Type Culture Collection (ATCC). PDA (2(pyridine-2-yldisulfanyl)ethyl acrylate) was synthesized with using aldrithiol-2 4.2.and Preparation mercaptoethanol of Melittin as Polymer described [23]. PDA was then copolymerized with poly (ethylene glycol) methacrylate at 1:1 ratio through AIBN initiated free radical polymerization to obtain PDA (2(pyridine-2-yldisulfanyl)ethyl acrylate) was synthesized with using aldrithiol-2 and poly[((2-(pyridin-2-yldisulfanyl)ethyl)acrylamide) co-[poly(ethylene glycol)]] (PDA-PEG). mercaptoethanol as described [23]. PDA was then copolymerized with poly (ethylene glycol) In order to produce PDA-PEG polymer with different amounts of amine and carboxylate methacrylate at 1:1 ratio through AIBN initiated free radical polymerization to obtain poly[((2- groups, cysteamine and 3-mercaptopropionic acid were conjugated to PDA-PEG at different ratios (pyridin-2-yldisulfanyl)ethyl)acrylamide) co-[poly(ethylene glycol)]] (PDA-PEG). (amine/carboxylate, molar ratio) through the thiol-disulfide exchange reaction (Figure2), and the In order to produce PDA-PEG polymer with different amounts of amine and carboxylate groups, cysteamine and 3-mercaptopropionic acid were conjugated to PDA-PEG at different ratios

Toxins 2020, 12, 582 10 of 13 resultant products were purified three times by precipitation in cold ether. Different feeding ratios between cysteamine and 3-mercaptopropionic acid yield polymers with different isoelectric points (IEP). LBA functionalization was carried out through EDC/NHS reaction. LBA was first activated with EDC/NHS in 0.1M MES buffer at pH 6.0 for 30 min, and then polymer PDA-PEG with modification of amine groups dissolved in PBS 7.4 at 20 mg/mL was added. The mixture was allowed to react overnight at room temperature. Free LBA was removed by repeated dialysis using Spectra/Por® dialysis tube (MWCO, 3.5 KDa) against dd H O for 48 h. Purified polymer was freeze-dried and stored at 20 C. 2 − ◦ The pyridine concentration in the polymer was quantitatively determined by measuring the UV absorbance at 375 nm after the cleavage by DTT. The concentration of pyridine was calculated based 1 1 on the extinction coefficient for pyridine-2-thione in DMSO at 375 nm (8080 M− cm− ). The molecular weight of polymers was determined by GPC Viscotek GPCmax VE 2001 GPC solvent/sample module equipped with Viscotek VE 3580 RI detector and 270 Dual Detector (Malvern Instruments, Worcestershire, United Kingdom) using THF as mobile phase, and the structure composition was determined by 1H-NMR. The success conjugation of 3-mercaptopropionic acid or cysteamine was confirmed by 1H-NMR (Bruker Avance III HD 300, Billerica, MA, USA) and with a UV-vis spectrometer (Beckman, DU650, Brea, CA, USA). The conjugation efficiency of LBA to PDA-PEG was quantified by a 2,4,6-trinitrobenzene sulfonic acid (TNBSA) assay using cysteamine hydrochloride as standard.

4.3. FRET Measurement The donor fluorescence dye Cy3-NHS was chemically conjugated with PDA-PEG, and the receptor fluorescence dye Sulfo-Cy5-NHS was conjugated with melittin following our published method [29]. Cy3 labeled polymer and Cy5 labeled melittin were mixed to prepared nanocomplex. Samples were loaded into Corning® 96 well black flat bottom plates and DTT was added to wells to cleave disulfide bonds. Samples were excited at 500 nm with the cutting off at 530 nm. The entire fluorescence spectrum (from 530 nm to 750 nm) of the complex treated prior to and after DTT was then recorded as a function of the time with the help of a SpectraMax M5 Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA, USA).

4.4. Hemolysis Experiment Rat whole blood was purchased from Bioreclamation (Hicksville, NY, USA). Red blood cells were washed with 150 mM NaCl and centrifuged at 300 rcf for 2 min until the supernatant became clear. The resulting red blood cells were suspended in PBS 7.4. Polymer complex or melittin was dispersed in different buffers at a series of concentrations from 0.1 µM to 5 µM. To mimic the physiological condition, nanocomplexes were dispersed in PBS 7.4 100 mM or PBS 5.0 100 mM. To mimic the intracellular condition, nanocomplexes were dispersed in 150 mM PBS + 10 mM GSH and incubated at 37 ◦C for 2 h. Melittin nanocomplex or melittin solution was incubated with above red blood cells for 1 h at 37 ◦C. The release of hemoglobin was quantified by measuring the absorbance at 405 nm of the supernatant in a microplate reader (ELX808, BioTek Instrument, Winooski, VT, USA) after centrifugation at 300 rcf. for 2 min. PBS and ddH2O treated RBCs were used as negative control and positive control, respectively. The degree of hemolytic was calculated as the following formula: hemolysis % = (Abs(test) Abs(PBS − negative control))/Abs(DI water positive control)) 100%. × 4.5. Cellular Uptake HCT 116 colon cancer and MCF-7 breast cancer cells were cultured in Dulbecco’s modification of Eagle’s medium (Corning, Glendale, AZ, USA) supplemented with 10% FBS and 1% penicillin–streptomycin (Life Technology, Grand Island, NY, USA) at 37 ◦C in 5% humidified CO2 atmosphere. Cells were seeded in 35 mm Petri dishes with a density of 200,000 cells/petri dish and allowed to grow overnight. Both non-targeting and targeting nano-complexes were applied to cells at the Cy3 concentration of 2 µM for 3 h. Cells were washed three times with PBS and fixed with 4% Toxins 2020, 12, 582 11 of 13 formaldehyde, followed by staining with Hoechst 33,342 for 15 min. The cells were imaged with a confocal laser scanning microscope (LSM 700, Zeiss, Oberkochen, Germany).

4.6. In Vitro Cytotoxicity HCT 116 and MCF-7 cells were seeded in 96-well plates at the density of 20,000 cells/well and incubated at 37 ◦C in 5% CO2 overnight. Melittin, non-targeted complex, or targeted complex were diluted with a complete medium at desired concentrations ranging from 0.1 to 10 µM. After 24 h of incubation, the media were replaced with 100 µL fresh media containing 1 mg/mL MTT reagent and incubated for 4 h. The formed MTT crystal was dissolved with a stop solution, and the final optical density of the medium was measured using a microplate reader (ELX808, BioTek Instrument, Inc., Winooski, VT, USA) at λ = 595 nm.

4.7. Tumor Growth Retardation Study Female nude mice were purchased from the Jackson lab and randomly assigned into 3 groups (3 mice/group). All animal experiments were conducted in accordance with NIH regulations and approved by the Institutional Animal Care and Use Committee of the University of South Carolina (Protocol No. 100851, approval date: 23 September 2015). Total 5 million HCT 116 cells suspended in PBS were subcutaneously injected into mice. When the tumor volume reached about 30 mm3, the mice were anesthetized with 3% of isoflurane and treated with LBA melittin nanoparticle at 2 mg/kg via intratumoral injection. For control groups, the same amount of buffer used in preparing complex or non-targeted nanoparticles was employed.

4.8. Statistics Analysis Data was expressed as mean standard deviation and was analyzed using Microsoft Excel ± and GraphPad Prism 6.0 (San Diego, CA, USA). Statistical analysis was performed using a one-way Analysis of Variation (ANOVA) among treatment groups and the control group. A p value < 0.05 was considered significant.

Author Contributions: Conceptualization, P.X.; methodology, B.C. and P.X.; software, B.C.; validation, B.C. and P.X.; formal analysis, B.C.; investigation, B.C.; writing—original draft preparation, B.C.; writing—review and editing, P.X.; supervision, P.X.; project administration, P.X.; funding acquisition, P.X. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the National Institutes of Health (1R21CA252360-01 and 1R01AG054839-01A1). Conflicts of Interest: The authors declare that there are no conflicts of interest.

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