Doxorobicin as cargo in a redox-responsive drug delivery system capped with water dispersible ZnS nanoparticles

In this work, we have prepared and investigated a redox-responsive drug delivery system (DDS) based on a porous carrier. Doxorubicin (DOX), a chemotherapy medication for treatment of different kinds of cancer, was used as a model drug in the study. DOX was loaded in ordered hexagonal mesoporous silica SBA-15, a nanoporous material with good biocompatibility, stability, large pore size and specific surface area (S_BET = 908 m² g⁻¹, V_P = 0.79 cm³ g⁻¹, d = 5.9 nm) and easy surface modification. To prepare the redox-responsive system, cystamine derivative ligands, with redox active disulphide linkers were grafted onto the surface of SBA-15. To ensure no significant premature release of DOX from the porous system, thioglycolic acid modified ZnS nanoparticles (ZnS–COOH NPs) were used as pore capping agents. The grafted redox-responsive cystamine derivative ligand containing disulphide linkers was bonded by a peptide bond to the thioglycolic acid groups of ZnS–COOH NPs, capping the pores. Once the disulphide bond was cleaved, the ZnS–COOH NPs caps were released and pores were opened to deliver the DOX cargo. The dithiol bond was cleavable by redox active molecules such as dithiothreitol (DTT) or glutathione, the concentration of which in cancer cells is 4 times higher than in healthy cells. The redox release of DOX was studied in two different media, physiological saline solution with DTT and saline without DTT. The prepared DDS proved the concept of redox responsive release. All samples were characterised by powder X-ray diffraction (XRD), transition electron microscopy (TEM), nitrogen adsorption/desorption at 77 K, Fourier-transform infrared spectroscopy (FTIR), thermal analysis and zeta potential measurements. The presence of semiconducting ZnS nanoparticle caps on the pore openings was detected by magnetic measurements using SQUID magnetometry showing that such cargo systems could be monitored using magnetic measurements which opens up the possibilities of using such drug delivery systems as theranostic agents.

Redox-responsive drug delivery system was studied. ZnS nanoparticles served as pore capping agent to prevent premature release of anticancer drug. Such cargo can be monitored by magnetic field which opens possibilities its use in theranostics.     

Highly-controllable drug release from core cross-linked singlet oxygen-responsive nanoparticles for cancer therapy

Highly-controllable release consisting of preventing unnecessary drug leakage at physiologically normal tissues and triggering sufficient drug release at tumor sites is the main aim of nanoparticle-based tumor therapy. Developing drug-conjugation strategies with covalent bonds in response to a characteristic stimulus, such as reactive oxygen species (ROS) generated by photodynamic therapy (PDT) has attracted much attention. ROS can not only cause cytotoxicity, but also trigger the cleavage of ROS-responsive linkers. Therefore, it is feasible to design a new model of controlled drug release via the breakage of ROS-responsive linkers and degradation of nanoparticles. The self-supply of the stimulus and highly-controllable drug release can be achieved by encapsulation of photosensitizer (PS) and chemotherapeutic drugs simultaneously without any support of tumor endogenous stimuli. Therefore, we used thioketal (TK) linkers as the responsive linkers due to their reaction with singlet oxygen (¹O₂, SO), a type of ROS. They were conjugated to the side groups of polyphosphoesters (PPE) via click chemistry to acquire the core cross-linked SO-responsive PPE nanoparticles poly(thioketal phosphoesters) (TK-PPE). TK-PPE coated with the photosensitizer chlorin e6 (Ce6) and chemotherapeutic drug doxorubicin (DOX) simultaneously were prepared and named as TK-PPE_Ce6&DOX. TK-PPE_Ce6&DOX kept stable due to the high stability of the TK-linkers in the normal physiological environment. With self-production of SO as the stimulating factor from the encapsulated Ce6, highly-controlled drug release was achieved. After incubation of tumor cells, 660 nm laser irradiation induced SO generation, resulting in the cleavage of TK-linkers and boosted-release of DOX. Highly-controllable drug release of TK-PPE_Ce6&DOX through self-production of stimulus increased antitumor efficacy, offering a promising avenue for clinical on-demand chemotherapy.

Core cross-linked singlet oxygen-responsive nanoparticle TK-PPE_Ce6&DOX could achieve highly-controllable drug release through self-production of SO as the stimulus to increase antitumor efficacy for cancer therapy.     

Inventing a facile method to construct Bombyx mori (B. mori) silk fibroin nanocapsules for drug delivery

Bombyx mori (B. mori) silk fibroin (SF) microcapsules have acted as a great candidate in delivering drugs. However, it is difficult to fabricate SF nanocapsules using the present layer-by-layer (LBL) technique. In addition, the current SF microcapsules have limits in loading negatively charged drugs. Here, we invent a novel LBL method by introducing silane (APTES) as a structure indicator to produce SF nanocapsules that can load drugs with negative or positive charge. LBL assembly was completed by alternately coating SF and APTES on the template of polystyrene (PS) nanospheres by electrostatic attraction. SF nanocapsules were obtained after removal of the PS templates. Zeta potential analysis proved LBL assembly was indeed driven by the interaction between negative charge of SF and positive charge of APTES. Fluorescence images and electric microscope images indicated that SF nanocapsules had a hollow and stable structure with diameter at nearly 250 nm. The highest encapsulation rate of DOX or Ce6 were up to 80% and 90%, respectively, indicating SF nanocapsules have a high loading capability for both cationic and anionic drugs. In vitro cell experiments proved the biocompatibility of SF nanocapsules and their burst drug release in response to acidic environment. Furthermore, chemotherapy and photodynamic therapy proved SF nanocapsules loaded with DOX or Ce6 had significant inhibition on tumor cells. Our results suggested that this LBL technique is a facile method for polymers with negative charge to fabricate nanocapsules for antitumor drug carrier.

A novel LBL method was proposed here by introducing silane to produce stable SF nanocapsules for better drug delivery.     

Therapeutic polymeric nanomedicine: GSH-responsive release promotes drug release for cancer synergistic chemotherapy

To obtain an efficient dual-drug release and enhance therapeutic efficiency for combination chemotherapy, a glutathione (GSH)-responsive therapeutic amphiphilic polyprodrug copolymer (mPEG-b-PCPT) is synthesized to load doxorubicin (DOX) via hydrophobic and π–π stacking interaction. In this nanomedicine system (mPEG-b-PCPT/DOX), the ratio of the two drugs can be easily modulated by changing the loading content of DOX. The in vitro drug release curves and laser confocal images suggested that the release of CPT and DOX is induced through a “release promotes release strategy”: after internalization into tumor cells, the disulfide bonds in the nanomedicine are cleaved by glutathione (GSH) in the cytoplasm and then lead to the release of CPT. Meanwhile, the disassembly of nanomedicine immediately promotes the co-release of DOX. The optimum dose ratio of CPT and DOX is evaluated via the combination index (CI) value using HepG-2 cells. The results of cell apoptosis and cell viability prove the better synergistic efficiency of the nanomedicine than free drugs at the optimum dose ratio of 1. Consequently, this stimuli-responsive synergistic chemotherapy system provides a direction for the fabrication of nanomedicines possessing promising potential in clinical trials.

In the GSH-responsive doxorubicin loading camptothecin prodrug nanomedicine, easy modulation of the dose ratio and controlled co-release were achieved, and the synergistic effect was significantly improved.     

RAFT polymerization mediated core–shell supramolecular assembly of PEGMA-co-stearic acid block co-polymer for efficient anticancer drug delivery

In this work, core–shell supramolecular assembly polymeric nano-architectures containing hydrophilic and hydrophobic segments were synthesized via reversible addition fragmentation chain transfer (RAFT) polymerization. Herein, polyethylene glycol methyl ether methacrylate (PEGMA), and stearic acid were used to synthesize the poly(PEGMA) homopolymer and stearyl ethyl methacrylate (SEMA), respectively. Then, PEGMA and SEMA were polymerized through controlled RAFT polymerization to obtain the final diblock copolymer, poly(PEGMA-co-SEMA) (BCP). Model anticancer drug, doxorubicin (DOX) was loaded on BCPs. Interestingly, efficient DOX release was observed at acidic pH, similar to the cancerous environment pH level. Significant cellular uptake of DOX loaded BCP50 (BCP50-DOX) was observed in MDA-MB-231 triple negative breast cancer cells and resulted in a 35 fold increase in anticancer activity against MDA MB-231 cells compared to free DOX. Scanning electron microscopy (SEM) imaging confirmed the apoptosis mediated cellular death. These core–shell supramolecular assembly polymeric nano-architectures may be an efficient anti-cancer drug delivery system in the future.

In this work, core–shell supramolecular assembly polymeric nano-architectures containing hydrophilic and hydrophobic segments were synthesized via reversible addition fragmentation chain transfer (RAFT) polymerization.     

Development and evaluation of a pH-responsive and water-soluble drug delivery system based on smart polymer coating of graphene nanosheets: an in silico study

The objective of this study is to develop a controlled and water-soluble delivery system for doxorubicin (DOX) based on the coating of graphene (G) with a smart polymer. A combination of polyethyleneimine (PEI) and G–DOX is investigated by performing density functional theory (DFT) calculations and molecular dynamics (MD) simulations. Several parameters have been employed to evaluate the effect of PEI on the adsorption and release mechanisms of DOX. The obtained results indicated that the binding energy of the drug molecule on G in the presence of PEI is enhanced by about 20% under neutral conditions, whereas the drug absorption becomes weaker in an acidic environment so that DOX could be separated from the carrier surface using near-infrared radiation (NIR). Based on the atom in molecule (AIM) theory, two hydrogen bonds with strengths of about −12.59 and −39.99 kJ mol⁻¹ have been established. Furthermore, evaluating the dynamic behavior of the designed systems in water solution shows that the polymer in physiological pH rapidly adsorbed on the drug–carrier complex. However, at acidic pH, it is quickly desorbed from the carrier surface and the G–DOX complex can be exposed to cancer cells. The obtained results of the present research may be used in future experimental work to design smart DDSs.

The objective of this study is to develop a controlled and water-soluble delivery system for doxorubicin (DOX) based on the coating of graphene (G) with a smart polymer.     

UCN–SiO2–GO: a core shell and conjugate system for controlling delivery of doxorubicin by 980 nm NIR pulse

Herein, graphene oxide (GO) has been attached with core–shell upconversion-silica (UCN–SiO₂) nanoparticles (NPs) to form a GO–UCN–SiO₂ hybrid nanocomposite and used for controlled drug delivery. The formation of the nanocomposite has been confirmed by various characterization techniques. To date, a number of reports are available on GO and its drug delivery applications, however, the synergic properties that arise due to the combination of GO, UCNPs and SiO₂ can be used for controlled drug delivery. New composite UCN@SiO₂–GO has been synthesized through a bio-conjugation approach and used for drug delivery applications to counter the lack of quantum efficiency of the upconversion process and control sustained release. A model anticancer drug (doxorubicin, DOX) has been loaded to UCNPs, UCN@SiO₂ NPs and the UCN@SiO₂–GO nanocomposite. The photosensitive release of DOX from the UCN@SiO₂–GO nanocomposite has been studied with 980 nm NIR laser excitation and the results obtained for UCNPs and UCN@SiO₂ NPs compared. It is revealed that the increase in the NIR laser irradiation time from 1 s to 30 s leads to an increase in the amount of DOX release in a controlled manner. In vitro studies using model cancer cell lines have been performed to check the effectiveness of our materials for controlled drug delivery and therapeutic applications. Obtained results showed that the designed UCN@SiO₂–GO nanocomposite can be used for controlled delivery based therapeutic applications and for cancer treatment.

A GO–UCN–SiO₂ hybrid nanocomposite for loading of doxorubicin and its use in in vitro efficiency for killing carcinoma cells.     

One-step dry synthesis of an iron based nano-biocomposite for controlled release of drugs

Bio-based drug carriers have gained significant importance in Control Drug Delivery Systems (CDDS). In the present work, a new iron-based magnetic nano bio-composite (nano-Fe-CNB) is developed in a one-step dry calcination process (solventless) using a seaweed-based biopolymer. The detailed analysis of the developed nano Fe-CNB is carried out using FE-SEM, HR-TEM, P-XRD, XPS, Raman spectroscopy, FTIR etc. and shows that nano-Fe-CNB consists of nanoparticles of 5–10 nm decorated on 7–8 nm thick 2-D graphitic carbon material. The impregnation of nano-Fe-CNB into the calcium alginate (CA) hydrogel beads is found to have good drug loading capacity as well as pH responsive control release behavior which is demonstrated using doxorubicin (DOX) as a model cancer drug. The drug loading experiments exhibit ∼94% loading of DOX and release shows ∼38% and ∼8% release of DOX at pH 5.4 and 7.4 respectively. The developed nano Fe-CNB facilitates strong electrostatic interactions with cationic DOX molecules at pH 7.4 and thereby restricts the release of the drug at physiological pH. However, at cancer cell pH (5.4), the interaction between the drug and nano-Fe-CNB reduces which facilitates more drug release at pH 5.4. Thus, the developed nano-biocomposite has the potential to reduce the undesired side effects associated with faster release of drugs.

Schematics for synthesis and application of magnetic nano-biocomposite for control release of DOX.     

DOX sensitized upconversion metal–organic frameworks for the pH responsive release and real-time detection of doxorubicin hydrochloride

Drug resistance is a major obstacle in cancer treatment, and designing a material that monitors real-time drug release remains a top priority. In this study, metal–organic frameworks doped with lanthanum and thulium were synthesized and then coated with aminated silica to form La/Tm-MOF@d-SiO₂ as a drug carrier. Doxorubicin hydrochloride (DOX) was selected as a drug model, and the drug loading and release were investigated. It was found that the release of DOX under acidic conditions reached an optimal level, indicating the pH-responsiveness of La/Tm-MOF@d-SiO₂. Under acidic conditions (pH = 5.8), upconversion fluorescence was generated after loading DOX on La/Tm-MOF@d-SiO₂. At pH = 5.8, the longer the drug released, the stronger the upconversion fluorescence. It was found that the upconversion fluorescence intensity is directly proportional to the amount of drug released; thus, the real-time monitoring of DOX release in tumor cells can be performed based on the upconversion fluorescence.

Drug resistance is a major obstacle in cancer treatment, and designing a material that monitors real-time drug release remains a top priority.     

Dual responsive PMEEECL–PAE block copolymers: a computational self-assembly and doxorubicin uptake study

The self-assembly behaviour of dual-responsive block copolymers and their ability to solubilize the anticancer drug doxorubicin (DOX) has been investigated using all-atom molecular dynamics (MD) simulations, MARTINI coarse-grained (CG) force field simulation and Scheutjens–Fleer self-consistent field (SCF) computations. These diblock copolymers, composed of poly{γ-2-[2-(2-methoxyethoxy)ethoxy]ethoxy-ε-caprolactone} (PMEEECL) and poly(β-amino ester) (PAE) are dual-responsive: the PMEEECL block is thermoresponsive (becomes insoluble above a certain temperature), while the PAE block is pH-responsive (becomes soluble below a certain pH). Three MEEECL₂₀–AE_M compositions with M = 5, 10, and 15, have been studied. All-atom MD simulations have been performed to calculate the coil-to-globule transition temperature (T_cg) of these copolymers and finding appropriate CG mapping for both PMEEECL–PAE and DOX. The output of the MARTINI CG simulations is in agreement with SCF predictions. The results show that DOX is solubilized with high efficiency (75–80%) at different concentrations inside the PMEEECL–PAE micelles, although, interestingly, the loading efficiency is reduced by increasing the drug concentration. The non-bonded interaction energy and the RDF between DOX and water beads confirm this result. Finally, MD simulations and SCF computations reveal that the responsive behaviour of PMEEECL–PAE self-assembled structures take place at temperature and pH ranges appropriate for drug delivery.

The self-assembly behaviour of dual-responsive block copolymers and their ability to solubilize the drug doxorubicin is demonstrated using molecular dynamics simulations, coarse-grained force field simulations and self-consistent field theory.     

Disulfide based prodrugs for cancer therapy

Advances in the tumor microenvironment have facilitated the development of novel anticancer drugs and delivery vehicles for improved therapeutic efficacy and decreased side effects. Disulfide bonds with unique chemical and biophysical properties can be used as cleavable linkers for the delivery of chemotherapeutic drugs. Accordingly, small molecule-, peptide-, polymer- and protein-based multifunctional prodrugs bearing cleavable disulfide bonds are well accepted in clinical settings. Herein, we first briefly introduce a number of prodrugs and divide them into three categories, namely, disulfide-containing small molecule conjugates, disulfide-containing cytotoxic agent–targeted fluorescent agent conjugates, and disulfide-containing cytotoxic agent–macromolecule conjugates. Then, we discuss the complex redox environment and the underlying mechanism of free drug release from disulfide based prodrugs in in vivo settings. Based on these insights, we analyze the impact of electronics, steric hindrance and substituent position of the disulfide linker on the extracellular stability and intracellular cleavage rate of disulfide containing prodrugs. Current challenges and future opportunities for the disulfide linker are provided at the end.

This review summarizes the progress in disulfide linker technology to balance extracellular stability and intracellular cleavage for optimized disulfide-containing prodrugs.     

A mitochondria-targeted delivery system of doxorubicin and evodiamine for the treatment of metastatic breast cancer

For mitochondria-targeted nano-drug delivery systems against cancer, effectively targeting and releasing the drug into mitochondria are the keys to improve the therapeutic effect. In this study, mitochondria-targeted and reduction-sensitive micelles were developed to co-deliver doxorubicin (DOX) and evodiamine (EVO) for the treatment of metastatic breast cancer. After entering cancer cells, the micelles first targeted mitochondria through triphenylphosphonium cations. Then, the disulfide bonds of the micelles were cleaved by GSH, and both DOX and EVO were released near the mitochondria. The released EVO subsequently destroyed the mitochondrial membrane, resulting in a large amount of DOX entering the mitochondria and improving the anti-tumor effect of DOX. These mitochondria-targeted and reduction-sensitive micelles loaded with doxorubicin and evodiamine showed significant inhibition of the tumor cell growth both in vitro and in vivo.

For mitochondria-targeted nano-drug delivery systems against cancer, effectively targeting and releasing the drug into mitochondria are the keys to improve the therapeutic effect.     

Effects of pore size on in vitro and in vivo anticancer efficacies of mesoporous silica nanoparticles

Mesoporous silica nanoparticles (MSN) have been widely applied for drug delivery systems. To investigate the effects of pore size on anticancer efficacies, MSN with different pore sizes but similar particle sizes and surface charges were synthesized via a microemulsion method. The pore structures of MSN were characterized by transmission electron microscopy (TEM), small-angle X-ray scattering (SAXS), and N₂ adsorption–desorption isotherms. Doxorubicin loaded MSN (DOX/MSN) were prepared and the minimum drug loading capacity was detected in DOX/MSN with a pore size of 2.3 nm (DOX/MSN2). DOX/MSN with a pore size of 8.2 nm (DOX/MSN8) showed a comparable drug loading amount in comparison with ones with a pore size of 5.4 nm (DOX/MSN5). In vitro drug release profiles showed that DOX/MSN5 could release DOX quickly and completely. Compared with DOX/MSN2 and DOX/MSN8, DOX/MSN5 showed the higher cellular uptake and nucleic concentration of DOX in QGY-7703 cells, which led to efficient cell-apoptosis induction and anti-proliferation effect, and thus the optimal in vivo anticancer activities. Taken together, these results highlighted the importance of pore size in anticancer efficacies, which would serve as a guideline in the rational design of MSN for cancer therapy.

MSN with suitable pore sizes achieved an outstanding performance for in vitro and in vivo antitumor efficacies.     

A porous reduced graphene oxide/chitosan-based nanocarrier as a delivery system of doxorubicin

Nowadays, the concept of drug transmission is an important topic in the field of drug delivery research. Drug delivery is the method or process of administering a pharmaceutical compound to achieve a therapeutic effect in humans or animals. In this study, we report the development of a novel platform for the loading and release of doxorubicin (DOX). It is based on porous reduced graphene oxide (prGO) nanosheets and chitosan (CS) biocompatible polymer, where prGO can be dispersed in chitosan through amide linkages. The loading and release of DOX on the CS-prGO nanocomposite were investigated by voltammetry, FE-SEM, and FTIR and UV-Vis spectroscopy methods. We showed that chitosan-modified prGO (CS-prGO) was an extremely efficient matrix. An efficient loading of DOX (86% at pH 7.00, time 3 h and initial concentration of 0.5 mg mL⁻¹) was observed on CS-prGO as compared to the case of prGO due to the presence of the –OH and –NH₂ groups of chitosan. At the normal physiological pH of 7.00, approximately 10% of DOX could be released from CS-prGO in a time span of 1 h; however, when exposed to pH 4.00, 25% of DOX was released in 1 h. After 20 h, 18% and 62% of DOX was released at pH 7.00 and 4.00, respectively. This illustrates the major benefits of the developed approach for biomedical applications.

Nowadays, the concept of drug transmission is an important topic in the field of drug delivery research.     

A nanoprobe for fluorescent monitoring of microRNA and targeted delivery of drugs

Multifunctional nano-materials that can be used to monitor the expression of specific biomarkers and serve as vehicles for controlled drug delivery are highly desirable. Herein, we report a new DNA-hybrid-gated core–shell upconversion nanoprobe (UCNP@MOF/DOX) for fluorescence analysis of microRNA-21 (miR-21), which also triggers the release of drug loaded in the probes for on-demand anti-cancer treatment. The nanoprobe is built on the merits of ultraviolet-visible light of upconversion nanoparticles (UCNPs) excited by near-infrared (NIR) and extraordinary loading capability of metal–organic frameworks (MOFs) for drug delivery. Controlled release of doxorubicin (DOX) from the nanoprobe by miR-21 underwent the following two-stage kinetics: a fast release stage specifically triggered by miR-21 and proportional to miR-21 concentration and a slow stage observed in both gated and ungated nanoprobes due to collapse of the UIO-66-NH₂ coatings via ligand exchange with phosphates. In addition, the nanoprobe showed good selectivity, a linear response towards miR-21 ranging from 4 nM to 500 nM, and a limit of detection in 4 nM, which precluded unintended payload leakage due to low-abundance endogenous miR-21 expression in normal cells. Moreover, based on a dual-targeted delivery system constituted by AS1411-mediated recognition and responsive release of DOX, a specific cytotoxic efficacy was observed in MCF-7 cells. The present work provides a smart and robust nanoprobe for real-time detection of miRNA and dual-responsive drug delivery in tumor cells.

A DNA-hybrid-gated core–shell upconversion nanoprobe is prepared for both fluorescent monitoring of miR-21 and on-demand delivery of DOX. It showed good selectivity towards miR-21 and demonstrated specific cytotoxic efficacy towards MCF-7 cells.     

Glutathione-depleting polymer delivering chlorin e6 for enhancing photodynamic therapy

The therapeutic effect of photodynamic therapy (PDT) is highly dependent on the intracellular production of reactive oxygen species (ROS). However, the ROS generated by photosensitizers can be consumed by the highly concentrated glutathione (GSH) in tumor cells, severely impairing the therapeutic effect of PDT. Herein, we synthesized a GSH-scavenging copolymer to deliver photosensitizer chlorin e6 (Ce6). The pyridyl disulfide groups, which have faster reactivity with the thiol groups of GSH than other disulfide groups, were grafted onto a hydrophobic block to encapsulate the Ce6. Under NIR irradiation, the Ce6 generated ROS to kill tumor cells, and the pyridyl disulfide groups depleted the GSH to prevent ROS consumption, which synergistically enhanced the therapeutic effect of PDT. In vitro and in vivo experiments confirmed the combinatory antitumor effect of Ce6-induced ROS generation and the pyridyl disulfide group-induced GSH depletion. Therefore, the pyridyl disulfide group-grafted amphiphilic copolymer provides a more efficient strategy for enhancing PDT and has promising potential for clinical application.

We report a novel GSH-depleting polymer based on a thiol–pyridine disulfide exchange reaction, with fast reactivity and high efficiency in GSH depletion that effectively promotes ROS accumulation and significantly enhances photodynamic therapy.     

Reduction-responsive molecularly imprinted nanogels for drug delivery applications

Degradable molecularly imprinted polymers (MIPs) with affinity for S-propranolol were prepared by the copolymerization of methacrylic acid as functional monomer and a disulfide-containing cross-linker, bis(2-methacryloyloxyethyl)disulfide (DSDMA), using bulk polymerization or high dilution polymerization for nanogels synthesis. The specificity and the selectivity of DSDMA-based molecularly imprinted polymers toward S-propranolol were studied in batch binding experiments, and their binding properties were compared to a traditional ethylene glycol dimethacrylate (EDMA)-based MIP. Nanosized MIPs prepared with DSDMA as crosslinker could be degraded into lower molecular weight linear polymers by cleaving the disulfide bonds and thus reversing cross-linking using different reducing agents (NaBH₄, DTT, GSH). Turbidity, viscosity, polymer size and IR-spectra were measured to study the polymer degradation. The loss of specific recognition and binding capacity of S-propranolol was also observed after MIP degradation. This phenomenon was applied to modulate the release properties of the MIP. In presence of GSH at its intracellular concentration, the S-propranolol release was higher, showing that these materials could potentially be applied as intracellular controlled drug delivery system.

Degradable molecularly imprinted polymers were prepared using redox sensitive cross-linkers and applied as intracellular drug delivery system to address the biocompatibility and cytotoxicity issues encountered with these synthetic polymers.     

Pluronic F127 self-assembled MoS2 nanocomposites as an effective glutathione responsive anticancer drug delivery system

In this study, bio-responsive polymeric MoS₂ nanocomposites were prepared for use as a drug carrier for cancer therapy. Herein, we report the synthesis and demonstrate the self-assembly of pluronic F127 (PF127) on a cystamine–glutathione–MoS₂ (CYS–GSH–MoS₂) system, which can be used for GSH-triggered drug release under biological reducing conditions. The reduction-sensitive disulfide bond containing CYS was incorporated between the amphiphilic copolymer PF127 and GSH–MoS₂ to achieve feasible drug release. Percent drug loading capacity and encapsulation efficiency were 51.3% and 56%, respectively. In addition, when the MoS₂–GSH–CYS–PF127 nanocomposite was incubated in a GSH environment, the morphology of the nanocomposite tended to change, ultimately leading to drug release. The drug-loaded PF127–CYS–GSH–MoS₂ polymeric nanocomposites efficiently released 52% of their drug content after 72 h of incubation in a GSH reduction environment. The HeLa cells treated with DOX loaded MoS₂–GSH–CYS–PF127 showed 38% toxicity at drug concentration of 40 μg, which indicated that the successfully released of drug from carrier and caused the cell death. Further, fluorescence microscopy images of HeLa cells revealed the potential behavior of the MoS₂–GSH–CYS–PF12 nanocomposite during the 2- and 4 h incubation periods; the nanocomposite was only found in the cytoplasm of HeLa cells. Interestingly, after 6 h of incubation, the drug was slowly released from the nanocomposite and could enter the nucleus as confirmed by fluorescence imaging of HeLa cells. Altogether, our synthesized PF127-coated MoS₂ nanocomposite could be effectively adopted in the near future as a GSH-sensitive drug carrier.

In this study, bio-responsive polymeric MoS₂ nanocomposites were prepared for use as a drug carrier for cancer therapy.     

Surface design and preparation of multi-functional magnetic nanoparticles for cancer cell targeting,therapy, and imaging

Recently, theranostic candidates based on superparamagnetic iron oxide nanoparticles (SPIONs) providing the combination of therapy and diagnosis have become one of the most promising system in cancer research. However, poor stability, premature drug release, lack of specific tumor cell targeting, and complicated multi-step synthesis processes still hinder them for potential clinical applications. In this research, the multi-functional magnetic nanoparticles (MNPs-DOX) were prepared via a simple assembly process for targeted delivery of doxorubicin (DOX) and enhanced magnetic resonance (MR) imaging detection. Firstly, the multi-functional copolymer coating, polyamidoamine (PAMAM), was designed and synthesized by Michael addition reaction, where N,N-bis(acryloyl)cystamine served as backbone linker, and DOX, dopamine (DA), and polyethylene glycol (PEG) acted as comonomers. The PAMAM was then directly assembled to the surface of SPIONs by the ligand exchange reaction with SPIONs forming the MNPs-DOX. The hydrophilic PEG moieties provide the nanoparticles with colloidal stability and good-dispersity in aqueous solution. Comparing with the quick release of free DOX, the drug release behavior of MNPs-DOX exhibited a sustained drug release. Because the chemical cleavage of disulfide in the polymer backbone, a high cumulative drug release up to 60% in GSH within 48 h was observed, rather than only 26% in PBS (pH 7.4) without GSH. The MR imaging detection experiment showed that the MNPs-DOX had an enhanced T₂ relaxivity of 126 mM⁻¹ S⁻¹ for MR imaging. The results of the cytotoxicity assays showed a remarkable killing effect of cancer cells by MNPs-DOX due to the FA tumor-targeting ligand, comparing with non-targeted drug molecules. All the results showed that the as prepared multi-functional magnetic nanoparticles may serve as a promising theranostic candidate for targeted anticancer drug delivery and efficient detection through MR imaging in medical application.

Multi-functional magnetic nanoparticles for targeted anticancer drug delivery and efficient MR imaging detection in theranostics.     

A biocompatible poly(amidoamine) (PAMAM) dendrimer octa-substituted with α-cyclodextrin towards the controlled release of doxorubicin hydrochloride from its ferrocenyl prodrug

Facile and efficient methods for the synthesis of the first poly(aminodamine) PAMAM G1.0 dendrimer octa-substituted with α-cyclodextrin and a novel ferrocenyl prodrug of doxorubicin hydrochloride are developed. This vector is non-toxic and can bind the designed ferrocenyl prodrug. It also shows a controlled drug release profile and high cytotoxicity against breast cancer cells (MCF-7), as elucidated by the in vitro biological studies performed with an innovative cell-on-a-chip microfluidic system.

A controlled release of doxorubicin hydrochloride from a novel nanoconjugate comprising PAMAM dendrimer octa-substituted with α-cyclodextrin and ferrocenyl prodrug is presented.

Poly(amidoamine) (PAMAM) dendrimers are of the highest interest to general medicinal chemistry.^1–3 These dendrimers show beneficial physicochemical or biological properties in comparison with the respective polyamine polymers, e.g., polyethylenimine (PEI). In general, the cytotoxicity of PAMAM dendrimers is lower in comparison to that of PEI, which is an important factor in terms of the development of non-viral drugs or gene delivery vectors. The unique shape of PAMAM dendrimers as well as the presence of highly reactive amino groups imply interesting possibilities towards the construction of novel systems dedicated to modern therapies in humans.In recent years, the chemistry and application of PAMAM dendrimer nanoconjugates with cyclodextrins (CDs) has drawn an unflagging interest.^4–7 CDs are the supramolecules formed of six, seven or eight d-glucose units, which are coupled via α-1,4-glycosidic bonds.^8,9 CDs form cup-shaped molecules. Their cavity is hydrophobic, whilst the exterior is hydrophilic. As a result, CDs show unique properties towards the formation of host–guest complexes with hydrophobic compounds, including drugs.^10–12 From the point of view of applied medicinal chemistry, the presence of CDs in the therapeutic system provides the possibility to release a drug in a controlled way. It is associated with the strategy of stepwise release of a drug from the inner cavity of CD. Furthermore, CDs increase the water solubility and/or biocompatibility of the drug delivery vector. The abovementioned features make CDs promising candidates for the decoration of PAMAM dendrimers. PAMAM dendrimers grafted with CD moieties can be used as versatile delivery agents. The uses of such macromolecular species cover the binding and release of various therapeutic species, including nucleic acids (e.g., siRNA or DNA)^13–16 or drugs (e.g., doxorubicin or sodium methotrexate).^6,17,18 These dendrimeric structures showed encouraging biological properties towards their use in medicinal chemistry, especially in terms of the design of novel anticancer agents. In some cases, additional structural motifs were introduced to these vectors, such as poly(ethylene glycol) (PEG) residues, towards providing specific biological or physicochemical properties.^18,19 The studies dealing with the application of PAMAM-CD architectures towards the construction of biosensors were also reported.^20,21 Furthermore, interesting studies on the solubilisation of highly hydrophobic fullerenes with PAMAM-CD-PEG vectors¹⁹ or cobaltocene-bridged PAMAM-CD dendrimers were also reported. These examples clearly elucidate the capabilities of PAMAM-CD nanoconjugates towards their use in modern applied chemistry, including nanomedicine.The use of ferrocene (Fc) in medicinal chemistry has been studied over the years.^22–31 Some of the reports deal with the synthesis of Fc-templated drugs^23,24 or prodrugs.^25–31 The latter concept is especially interesting from the point of view of applied medicinal chemistry, since prodrug technology may improve the biocompatibility and/or bioaccessibility of a drug.^32–34 However, the reports dealing with the formation of Fc-based prodrugs are still sparse; they cover, e.g., the synthesis of Fc-functionalized nucleobases³¹ or synthesis and biological evaluation of the prodrugs bearing Fc and boronic acid moieties.^25,26 Interestingly, Fc is known for the formation of stable host–guest inclusion complexes with CD.^27,28 Fc is not soluble in water, thus, it is not released from its complex with CD in an aqueous medium. Fc release can be only achieved via a redox process (ferrocenyl cation does not form stable inclusion complexes) and the employment of this concept can be indeed found in the literature.^29,30 Thus, Fc can also be employed as the building block for macromolecular therapeutic systems, including self-assembling drug delivery systems.^35–38 An interesting example is the formation of a pH-responsive supramolecular system for controlled drug release, which is based on the self-assembly of the Fc-PEG conjugate and β-cyclodextrin-functionalized doxorubicin hydrochloride.³⁵ The drug in this system, that is doxorubicin hydrochloride (DOX*HCl), was released by means of an oxidant-dependent process. This system showed promising biological features towards cancer treatments. In fact, DOX*HCl is commonly the first and/or best choice drug for the treatment of various cancers, including breast or lung cancer.^39–41In pursuit of the design of novel anticancer agents, herein, we present efficient and facile methods for the preparation of the first PAMAM G1.0 dendrimer octa-substituted with α-cyclodextrin (octa-αCD-PAMAM) and a novel DOX*HCl prodrug, namely ferrocenyl ester of doxorubicin hydrochloride (Fc–COO–DOX*HCl). Octa-αCD-PAMAM is non-toxic and has the property to bind Fc–COO–DOX*HCl. The in vitro studies revealed encouraging biological features of the designed nanoconjugate, namely controlled drug release behavior and high cytotoxicity against breast cancer cell line (MCF-7). In vitro biological assays were performed with an innovative cell-on-a-chip microfluidic system. We anticipate our findings will further stimulate the progress in medicinal chemistry with the use of macromolecular therapeutic systems exhibiting a controlled drug release profile.The procedure for the synthesis of octa-αCD-PAMAM (3) is presented in Fig. 1. In general, this derivative of PAMAM G1.0 (1) was obtained in good yield (80%) by means of a reductive amination approach with the use of α-cyclodextrin monoaldehyde (αCD-CHO; 2). The reaction occurred between each of the eight terminals, primary amino groups of 1, and formyl moieties of 2. In the first step of the reaction, imine-bonds were formed and then they were reduced to CH₂NH₂ linkages by means of the treatment with sodium triacetoxyborohydride.⁴² The obtained octa-αCD-PAMAM (3) was characterized with NMR and FT-IR spectroscopies, as well as with ESI-MS.⁴³ It is noteworthy that elemental analysis and ESI-MS experiment ultimately confirmed the introduction of eight αCD residues to one molecule of PAMAM G1.0; the calculated and found data were highly consistent. It means that the herein developed methodology enables the full functionalization of PAMAM''s terminal amino groups with biocompatible, αCD residues.Open in a separate windowFig. 1Synthesis of octa-αCD-PAMAM (3).The ferrocenyl ester of DOX*HCl (Fc–COO–DOX*HCl; 5) was obtained by means of the treatment of DOX*HCl with ferrocenecarboxylic acid (Fc-COOH; 4). The synthetic scheme is presented in Fig. 2. This process was based on the carbodiimide-mediated ester bond formation reaction (Steglich esterification) with the inclusion of a carboxyl group of 4 and the terminal CH₂OH moiety of DOX*HCl.⁴² It is worth noting that no reaction occurred between the amino group of DOX*HCl since this moiety remained in the form of hydrochloride during all the reaction and purification steps (no alkaline conditions were applied in our synthesis). Thus, in our methodology native DOX*HCl can be used, without the need for amino group protection⁴⁴ or use of enzymatic process.⁴⁵ Combination of NMR and FT-IR spectroscopies, as well as high-resolution mass spectrometry (HRMS) confirmed the formation of pure Fc–COO–DOX*HCl (5), a novel DOX*HCl prodrug, which bears the ferrocenyl moiety.⁴³Open in a separate windowFig. 2Synthesis of Fc–COO–DOX*HCl (5).With both octa-αCD-PAMAM (3) and Fc–COO–DOX*HCl (5) at hand, we began to merge their chemistries (Fig. 3). Our concept originated from the following facts. Fc is known for its capability to form very stable complexes with αCD.^27,28 αCD can accommodate one Fc residue, since the width of the inner cavity of αCD equals to 5.7 Å, whilst its depth is 7.8 Å. On the other hand, DOX*HCl molecule is too big to be effectively complexed inside the inner cavity of αCD; for this purpose, a larger CD should be used, such as β-cyclodextrin (width of inner cavity 7.8 Å) or γ-cyclodextrin (width of inner cavity 8.8 Å).^46–48 Therefore, in our system, Fc-mediated complexation with Fc–COO–DOX*HCl (5) and αCD units of octa-αCD-PAMAM (3) occurs. We have successfully obtained the desired nanoconjugate {Fc–COO–DOX*HCl}@{octa-αCD-PAMAM} (6) in quantitative yields using a combination of solution and lyophilisation methodology.⁴² FT-IR spectroscopy suggested the anticipated Fc-oriented complexation for this nanoconjugate, since no absorption bands coming from Fc moiety of 5 were observed in the spectrum of nanoconjugate 6, whilst absorption bands coming from DOX*HCl were found.⁴⁹ Importantly, ESI-MS and elemental analysis confirmed the formation of the desired nanoconjugate 6; the calculated and obtained data were highly consistent.⁴³ Additionally, we further studied the complex formation phenomenon with NMR techniques. At first, the Fc-oriented complexation was tracked with ¹H–¹H ROESY NMR.²⁵ The ¹H–¹H ROESY NMR spectrum of 6 featured the cross-correlations between Fc''s cyclopentadienyl signals (H_Cp) and H-3, H-5 inner protons of α-CD (Fig. 4). It was ascribed to the inclusion of Fc inside α-CD''s inner cavity. It stands for the successful formation of inclusion complexes between guest 5 and α-CD units of 3. Secondly, the results of ¹H DOSY NMR analysis suggested the formation of a single host–guest system. ¹H DOSY NMR technique involves the measurement of the diffusion coefficient of the compounds forming a sample and is a powerful and versatile NMR method for the analyses of the supramolecular systems, including host-guest complexes.^25,50,51 The ¹H DOSY NMR spectrum of nanoconjugate 6 showed one diffusion coefficient value (0.358 10⁻¹⁰ m² s⁻¹).^52b Thus, we hypothesized that a single host-guest system might have been formed. In other words, we claim that neither unbound 5 nor other dendrimeric structures (i.e., bearing less than eight complexed molecules of 5) were found in the sample. To further support this hypothesis, ¹H DOSY NMR spectra in the same solvent were acquired for native host 3 and guest 5.^52a Both of these showed higher diffusion coefficient values than the resultant nanoconjugate 6. As expected, the diffusion coefficient value for 5 (3.699 10⁻¹⁰ m² s⁻¹) was found to be higher than that for 3 (0.746 10⁻¹⁰ m² s⁻¹; this is because 3 is much bigger than 5). This clear difference in the diffusion coefficient values between the molecules forming the system (3, 5) and their resultant inclusion complex 6 support our claim on the host–guest chemistry behaviour for the studied system.⁵² Finally, UV-Vis spectroscopy was applied to provide an insight into the stoichiometry of the host-guest complexes of 3 and 5.²⁵ The UV-Vis spectra of guest 5 featured an increase in the absorbance in the presence of host 3, as well as some slight blue shift behaviour.^52b These features were ascribed to the inclusion phenomenon. This change differed between the samples that enabled the estimation of complex stoichiometry. The complex stoichiometry was estimated on the basis of Job''s plot analysis.²⁵ The estimated stoichiometry was found to be 1 : 8 (host : guest);^52b this conclusion supported the outcomes from the ESI-MS experiment and is highly consistent with other above-presented supramolecular analyses. All these important features mentioned above mean that the herein developed methodology enables full “blocking” of αCD''s cavities with ferrocenyl units of DOX*HCl prodrug 5 by means of the formation of Fc-oriented complexes.Open in a separate windowFig. 3Synthesis of Fc–COO–DOX*HCl (5). For the structures of 3 and 5, see Fig. 1 and ​and22.Open in a separate windowFig. 4The 4.30–3.45 ppm inset of the ¹H–¹H ROESY NMR (DMSO-d₆: D₂O = 1 : 1 v/v, 500 MHz) spectrum of nanoconjugate {Fc–COO–DOX*HCl}@{octa-αCD-PAMAM} (6) presenting the crucial cross-correlations standing for the inclusion phenomenon (these cross-correlations are marked in blue). The graphical representation of the complex is also shown.We envision that DOX*HCl might be released from nanoconjugate 6 under acidic conditions. Our hypothesis was based on two facts. Firstly, DOX*HCl is bound to this nanoconjugate in the form of its ferrocenyl prodrug (ester bond) by means of Fc-oriented complexation. Ester bonds are known for their prospective use in prodrug technologies.^32–34 Secondly, the pH of cancer cells was found to be acidic (pH 4–6).^53–55 It gives the possibility of a controlled drug release at the therapeutic target (cancer cell environment). In order to examine the possibility of DOX*HCl release from nanoconjugate 6 and the profile of this release, in vitro controlled drug release trials at pH 4.7 were performed.⁵⁶ The DOX*HCl release curve is presented in Fig. 5a, blue curve. This curve resembles the characteristic controlled drug release profile. It means that DOX*HCl release from nanoconjugate 6 was stepwise. This controlled release was ascribed to the hydrolysis of ester bonds between Fc and DOX*HCl parts of compound 5 complexed inside αCD units within nanoconjugate 6. The cumulative release of DOX*HCl after 24 hours equalled to ca. 78% and the final cumulative release (after 72 hours) was found to be ca. 87%. The first, relatively fast, DOX*HCl release segment up to ca. 12 h was ascribed to the release of DOX*HCl molecules that were close to the dendrimer–buffer interface.⁵⁷ Subsequently, the cumulative release of DOX*HCl increased gradually with the contact time. The plateau segment was achieved between 48 h and 72 h. This high total cumulative release value at a rationally short time constitutes a good starting point for the design of novel macromolecular therapeutics exhibiting a controlled drug release profile. For comparison, drug release trials were also performed at pH 7.4 (physiological pH; Fig. 5a, red curve). No significant drug release was found in this environment (cumulative DOX release was lower than ca. 1.5%, which means that in practise no compound, neither 5 nor any of its subpart (e.g., DOX), was released from 6). This finding means that (i) for our system simple equilibrium displacement during the dialysis did not take place, which confirms that the release of the drug is driven by acidic pH (hydrolysis of an ester bond), (ii) no unbound 5 was present in nanoconjugate 6.Open in a separate windowFig. 5(a) DOX*HCl release curves from nanoconjugate 6 at pH 4.7 and pH 7.4; (b) MCF-7 cell viability after treatment during long-term spheroid culture.Encouraged by the above-presented results, we estimated the cytotoxicity profile of the designed nanoconjugate 6 against breast cancer (MCF-7) spheroids. These studies were performed using an innovative cell-on-a-chip microfluidic system. Cell-on-a-chip are miniature, microfluidic devices that contain in vitro cell cultures under flow conditions that simulate physiology at the tissue level.⁵⁸ Unlike conventional in vitro cell culture methods, microfluidic-based cell cultures to a greater extent reproduce the in vivo conditions. It is associated with the combination of surfaces mimicking extracellular matrix geometries and microfluidic channels that regulate fluid transport (nutrients important for cells).⁵⁹ In our research, we used the innovative microfluidic device for long-term three-dimensional (3D) spheroid cell culture.⁶⁰ The use of three-dimensional cell contact and the flow conditions in a single device allowed more than standard two-dimensional cell cultures to reproduce the in vivo environment of breast cancer. Moreover, this device allowed us to perform quick and precise microscopic and fluorescence analysis after the drug treatment. The microscopic analysis involved the observation of morphological spheroid changes (in single, always the same spheroid) while fluorescence analysis involved the evaluation of the metabolic activity of spheroids (their viability). The results of biological assays are shown in Fig. 5b.⁶¹ At first, the cytotoxicity profile of octa-αCD-PAMAM (3) was estimated. This dendrimeric vector in each tested concentration was found to be non-toxic, which is beneficial in terms of its use as a drug delivery vector. The same situation was observed with free DOX*HCl. On the other hand, nanoconjugate 6 showed different cytotoxicity profiles; nanoconjugate 6 was found to be highly toxic to breast cancer cells. Cell viability after 72 h equated to ca. 40%. In comparison, this viability for octa-αCD-PAMAM (3) and free DOX*HCl (50 μg mL⁻¹, 72 h) was ca. 95% and ca. 81%, respectively. During our studies, we also observed that higher concentrations of the tested substances were less toxic to breast cancer cells. This can be related to the defense mechanism of cancer tumors; cancer tumors recognize higher concentrations of toxic substances and do not absorb them from the external environment.⁶² In addition, higher cell viability may be associated with the stimulation of cell proliferation after using higher concentrations of octa-αCD-PAMAM. We observed that the free drug carrier at higher concentrations increases the viability of MCF-7 cells (Fig. 5b). Similarly, the MCF-7 viability after treatment with a higher concentration of nanoconjugate 6 was also higher.