Energy-Dependent Endocytosis Is Responsible for Skin Penetration of Formulations Based on a Combination of Indomethacin Nanoparticles and l-Menthol in Rat and Göttingen Minipig

We previously designed a Carbopol gel formulation (N-IND/MEN) based on a combination of indomethacin solid nanoparticles (IND-NPs) and l-menthol, and we reported that the N-IND/MEN showed high transdermal penetration. However, the detailed mechanism for transdermal penetration of IND-NPs was not clearly defined. In this study, we investigated whether endocytosis in the skin tissue of rat and Göttingen minipig is related to the transdermal penetration of IND-NPs using pharmacological inhibitors of endocytosis. The pharmacological inhibitors used in this study are as follows: 54 µM nystatin, a caveolae-mediated endocytosis (CavME) inhibitor; 40 µM dynasore, a clathrin-mediated endocytosis (CME) inhibitor; and 2 µM rottlerin, a micropinocytosis (MP) inhibitor. The N-IND/MEN was prepared by a bead mill method, and the particle size of solid indomethacin was 79–216 nm. In both rat and Göttingen minipig skin, skin penetration of approximately 80% IND-NPs was limited by the stratum corneum (SC), although the penetration of SC was improved by the combination of l-menthol. On the other hand, the treatment of nystatin and dynasore decreased the transdermal penetration of indomethacin in rats and Göttingen minipigs treated with N-IND/MEN. Moreover, in addition to nystatin and dynasore, rottlerin attenuated the transdermal penetration of IND-NPs in the Göttingen minipigs’ skin. In conclusion, we found that l-menthol enhanced the SC penetration of IND-NPs. In addition, this study suggests that the SC-passed IND-NPs are absorbed into the skin tissue by energy-dependent endocytosis (CavME, CME, and/or MP pathways) on the epidermis under the SC, resulting in an enhancement in transdermal penetration of IND-NPs. These findings provide significant information for the design of nanomedicines in transdermal formulations.     

pH-responsive PEGylated nanogel containing platinum nanoparticles: Application to on–off regulation of catalytic activity for reactive oxygen species

Synthesis of pH-responsive PEGylated nanogels platinum nanoparticles (PtNPs: <2 nm) was successfully carried out through the reduction of K₂PtCl₆ within the PEGylated nanogels constructed from cross-linked poly[2-(N,N-diethylamino)ethyl methacrylate] (PDEAMA) core and tethered PEG chains. The resulting PEGylated nanogels containing PtNPs showed significant catalytic activity for reactive oxygen species (ROS) in response to skin-environmental pH (acid), whereas the almost no catalytic activity for ROS was observed at physiological pH due to the volume phase transition of the PDEAMA gel core. Thus, pH-responsive and PEGylated nanogel containing PtNPs can be utilized to the skin-specific ROS-scavengers for the skin aging.     

Nanogels comprising reduction-cleavable polymers for glutathione-induced intracellular curcumin delivery

In this study, we prepared nanogels of a disulfide-cleavable polymer via polyionic complexation and genipin cross-linking and evaluated their reduction-triggered intracellular curcumin (Cur) delivery. These nanogels were stable at physiological conditions due to the formation of genipin cross-linking and helical PLL/PDC complexes and would swell/dissociate at acidic and reductive conditions due to the dissociation of PLL/PDC complexes and cleaving of disulfide bonds. The cellular uptake and intracellular release of Cur-loaded nanogels were demonstrated by tracking the fluorescent Cur and in vitro drug release studies, confirming the triggered release of Cur at acidic and reductive microenvironments in cells. The MTT, TUNEL staining, and Caspase-3 activity assays showed that the Cur-loaded nanogels exhibited higher cellular proliferation inhibition toward U-87 MG cells than free Cur, whereas the blank nanogels exhibited low cytoxicity. The results highlight the potential of functional nanogels prepared by polyionic complexation and cross-linking as a smart nanocarrier for drug delivery.     

壳聚糖季铵盐修饰的辅酶Q10纳米结构脂质载体的制备及表征

为了增强运载辅酶Q10的纳米结构脂质载体(CoQ10-NLC)的透皮效果,采用促渗剂——壳聚糖季铵盐(QCS)对CoQ10-NLC进行了表面修饰,并结合QCS分子链的自聚集行为,明确了壳聚糖季铵盐修饰的包载辅酶Q10的纳米脂质体(QCS-CoQ10-NLC)的形成机制,得到了粒径在500 nm左右的脂质载体。进一步通过体外透皮实验,考察了QCS-CoQ10-NLC的透皮吸收效果。结果显示,经质量分数0.5%的QCS修饰后,脂质载体可将CoQ10在皮肤中的渗透总量由1.36μg/cm~2显著增加至5.14μg/cm~2。     

Elastic and Ultradeformable Liposomes for Transdermal Delivery of Active Pharmaceutical Ingredients (APIs)

Administration of active pharmaceutical ingredients (APIs) through the skin, by means of topical drug delivery systems, is an advanced therapeutic approach. As the skin is the largest organ of the human body, primarily acting as a natural protective barrier against permeation of xenobiotics, specific strategies to overcome this barrier are needed. Liposomes are nanometric-sized delivery systems composed of phospholipids, which are key components of cell membranes, making liposomes well tolerated and devoid of toxicity. As their lipid compositions are similar to those of the skin, liposomes are used as topical, dermal, and transdermal delivery systems. However, permeation of the first generation of liposomes through the skin posed some limitations; thus, a second generation of liposomes has emerged, overcoming permeability problems. Various mechanisms of permeation/penetration of elastic/ultra-deformable liposomes into the skin have been proposed; however, debate continues on their extent/mechanisms of permeation/penetration. In vivo bioavailability of an API administered in the form of ultra-deformable liposomes is similar to the bioavailability achieved when the same API is administered in the form of a solution by subcutaneous or epi-cutaneous injection, which demonstrates their applicability in transdermal drug delivery.     

The Anticancer Potential of Doxycycline and Minocycline—A Comparative Study on Amelanotic Melanoma Cell Lines

Malignant melanoma is still a serious medical problem. Relatively high mortality, a still-growing number of newly diagnosed cases, and insufficiently effective methods of therapy necessitate melanoma research. Tetracyclines are compounds with pleiotropic pharmacological properties. Previously published studies on melanotic melanoma cells ascertained that minocycline and doxycycline exerted an anti-melanoma effect. The purpose of the study was to assess the anti-melanoma potential and mechanisms of action of minocycline and doxycycline using A375 and C32 human amelanotic melanoma cell lines. The obtained results indicate that the tested drugs inhibited proliferation, decreased cell viability, and induced apoptosis in amelanotic melanoma cells. The treatment caused changes in the cell cycle profile and decreased the intracellular level of reduced thiols and mitochondrial membrane potential. The exposure of A375 and C32 cells to minocycline and doxycycline triggered the release of cytochrome c and activated initiator and effector caspases. The anti-melanoma effect of analyzed drugs appeared to be related to the up-regulation of ERK1/2 and MITF. Moreover, it was noticed that minocycline and doxycycline increased the level of LC3A/B, an autophagy marker, in A375 cells. In summary, the study showed the pleiotropic anti-cancer action of minocycline and doxycycline against amelanotic melanoma cells. Considering all results, it could be concluded that doxycycline was a more potent drug than minocycline.     

Multi stimuli-responsive HPC-PAA/Eu(AA)3Phen complex nanogels and their decoration with 3-APBA for detecting glucose

In this paper, novel multi stimuli-responsive complex nanogels of hydroxypropylcellulose (HPC)-PAA/Eu(acrylic acid [AA])₃Phen were synthesized by radical polymerization method with HPC, rare earth complex (Eu(AA)₃Phen) and AA as raw materials, and the 3-aminophenylboronic acid (3-APBA) decorated nanogels (HPC-PAAPBA/Eu(AA)₃Phen) were fabricated via condensation of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride with 3-APBA. The microstructure, morphology and diameter of the nanogel were characterized by Fourier transform infrared spectrometer, UV–Visible spectrophotometer, transmission electron microscope, dynamic light scattering. Moreover, the fluorescence responsive performance of complex nanogels to temperature, pH value and glucose concentration was investigated by photoluminescence spectroscopy. The results showed that both of the as-prepared nanogels were uniform in size and had good monodispersity. The temperature and pH value had significant effects on the particle size and the fluorescence emission intensity of the HPC-PAA/Eu(AA)₃Phen complex nanogels; after decoration with 3-APBA, the obtained HPC-PAAPBA/Eu(AA)₃Phen nanogels showed excellent stimulus-response to glucose concentration.     

Dieckol Inhibits Autophagic Flux and Induces Apoptotic Cell Death in A375 Human Melanoma Cells via Lysosomal Dysfunction and Mitochondrial Membrane Impairment

Dieckol is a natural brown algal-derived polyphenol and its cytotoxic potential against various types of cancer cells has been studied. However, the effects of dieckol on autophagy in cancer cells remain unknown. Here, we show that dieckol inhibits the growth of A375 human melanoma cells by inducing apoptotic cell death, which is associated with lysosomal dysfunction and the inhibition of autophagic flux. Dieckol induces autophagosome accumulation by inhibiting autophagosome-lysosome fusion. Moreover, dieckol not only triggers lysosomal membrane permeabilization, followed by an increase in lysosomal pH and the inactivation of cathepsin B and D, but also causes the loss of mitochondrial membrane potential. Importantly, a cathepsin D inhibitor partially relieved dieckol-induced mitochondrial membrane impairment and caspase-mediated apoptosis. Collectively, our findings indicate that dieckol is a novel autophagy inhibitor that induces apoptosis-mediated cell death via lysosomal dysfunction and mitochondrial membrane impairment in A375 human melanoma cells. This suggests the novel potential value of dieckol as a chemotherapeutic drug candidate for melanoma treatment.     

透皮给药系统的研究进展

介绍了透皮给药系统的特点、剂型、促渗技术和体内外研究模型,为透皮给药系统的研究提供参考。重点查阅了国外有关透皮给药系统的相关文献并进行分析总结。随着新材料、新技术、新设备的不断发展,促渗方法取得了很大的进步,透皮给药系统具有广阔的发展前景。     

海蜇、牛骨和鳕鱼皮胶原肽的透皮吸收性能研究

以海蜇、牛骨和鳕鱼皮胶原为原料,采用酶解制备了不同分子量(1 kDa和1~3 kDa)的6种胶原肽。利用透皮扩散试验仪,考察了时间、分子量及质量浓度对胶原肽透皮吸收性的影响,通过荧光显微镜观察了异硫氰酸荧光素(FITC)标记的胶原肽对小鼠皮肤的透皮吸收过程。结果表明,6种胶原肽的质量浓度均为10 g/L时,其在24 h时透皮吸收能力由大到小依次为:1 kDa鳕鱼皮胶原肽、1 kDa牛骨胶原肽、1 kDa海蜇胶原肽、1~3 kDa海蜇胶原肽、1~3kDa牛骨胶原肽和1~3 kDa鳕鱼皮胶原肽;1 kDa胶原肽透皮吸收能力优于1~3 kDa胶原肽。牛骨和海蜇胶原肽的质量浓度越大,透皮吸收性越好,而鳕鱼皮胶原肽则相反。3种来源胶原肽单位面积累积透过量均随时间延长而增加,分子量越大透皮吸收性能越差。     

聚甲基丙烯酸缩水甘油酯疏水纳凝胶的制备与表征

采用乳液聚合法制备了聚甲基丙烯酸缩水甘油酯疏水纳凝胶。通过透射电子显微镜、傅里叶红外光谱和动态激光光散射等测试对其结构和形貌进行了表征,对疏水纳凝胶形成过程规律和对温度、pH和时间稳定性进行了考察,并探究了单体浓度、交联剂含量和乳化剂浓度对纳凝胶粒径的影响规律。结果表明：所得疏水纳凝胶具有粒径均一、分散稳定和溶胀性能好等优点。疏水纳凝胶粒径在一定温度和pH范围内无变化,在30天内粒径从76nm增至116nm,zeta电位随温度升高而增加,其值稳定在-90~-30mV之间,说明是一种较为稳定的凝胶体系。在实验范围内,纳凝胶粒径随单体甲基丙烯酸缩水甘油酯和交联剂二甲基丙烯酸乙二醇酯浓度的增加从80nm增至250nm,随乳化剂浓度增加从230nm降至60nm。     

大豆7S球蛋白与葡聚糖共价键合纳米凝胶的制备与表征

基于蛋白质与多糖的Maillard反应与自组装制备一种新型的绿色的具备核壳结构的纳米凝胶。利用干热反应制备大豆7S球蛋白与葡聚糖的共价接枝物（soy β-conglycinin-dextran conjugates,SDC）,通过对SDC热处理使其自组装成大豆7S球蛋白-葡聚糖纳米凝胶（soy β-conglycinin-dextran nanogels,SDN）,对其形貌、结构及性质进行分析,并利用多糖的空间位阻效应抑制蛋白质宏观过度聚集的理论指导,探讨尺寸均一SDN的形成机制。形貌学与zeta电位分析表明SDN为具备核壳结构的球状粒子,其外壳由亲水性的葡聚糖构成,内核由凝胶化的蛋白构成;表面疏水性分析表明内核蛋白的三级结构发生转变,疏水基团暴露,从而形成多个疏水空腔;稳定性分析表明SDN具有高度的pH稳定性与储存稳定性,对疏水活性物质的输送载体构建具有重要的借鉴意义。     

Synthesis and evaluation of luliconazole loaded biodegradable nanogels prepared by pH-responsive Poly (acrylic acid) grafted Sodium Carboxymethyl Cellulose using amine based cross linker for topical targeting: In vitro and Ex vivo assessment

ABSTRACT

Fungal infection in immuno compromised patients causes skin syndromes and problems. At Present, innovative alternatives are required to cure skin disorders and infections. Luliconazole is a novel, broad spectrum, imidazole antifungal agent. The purpose of this study was to develop biodegradable, pH responsive, chemically cross-linked and Poly (acrylic acid) grafted sodium carboxymethyl cellulose nanogels. Nanogels had been synthesized to evaluate its applicability as an effective carrier of luliconazole for topical (skin) targeting. Chemically cross-linked sodium carboxymethyl cellulose-grafted-Poly acrylic acid (NaCMC-g-PAA) was synthesized from acrylic acid and sodium carboxymethyl cellulose using N, N’-methylene bisacrylamide (cross-linker) and potassium persulfate (initiator) using free radical polymerization. Variation of reaction parameters such as pH, cross linker, initiator and temperature has been used to optimize the best one. The developed nanogels reveal significant pH sensitive drug releasing behavior. NaCMC-g-PAA nanogels has been characterized using various physicochemical characterization techniques. Nanogels characteristics were evaluated through the In vitro drug release, Ex vivo permeation study, Nuclear magnetic resonance spectroscopy, Fourier Transform Infrared Spectroscopy, Field Emission Scanning Electron Microscope, Stability Study and antifungal activity. All batches were characterized for particle size analysis and ranged from 78.82 nm to 190 nm. The viscosity of developed nanogels was found to be 5941 cps. It was observed that the developed drug-loaded NaCMC-g-PAA nanogels were more effective in killing the fungus. Consequently, Nanogels incorporated with luliconazole could be a new approach with improved antifungal activity and increased topical delivery for a drug with poor aqueous solubility rather than coarse drug-containing cream.     

A versatile strategy to construct surface functionalized pH/redox dual stimuli-responsive nanogels for targeted drug delivery

Developing facile, green, and efficient synthetic methodology for preparing surface-functionalized nanogels has attracted tremendous attention in the field of polymer chemistry and biomaterials engineering. Herein, a series of “clickable” P(MAA-PMA) nanogels with reactive alkyne groups were conveniently prepared by “one-pot” reflux-precipitation polymerization (RPP), their particle sizes and zeta potentials are solid content-dependent. Using P(MAA-PMA)-1 nanogels as the model, the physicochemical properties including particle size, surface potential, morphology, solution stability, and pH/redox responsiveness were characterized in detail. Moreover, the pH/redox dual-responsive drug release feature/kinetics and parameters were analyzed using Korsmeyer–Peppas model and Gallagher–Corrigan model. Then the surface of nanogels was facilely modified with various thiol-containing functional ligands via thiol-yne photo click reaction. As targeted drug carriers, doxorubicin (DOX)-loaded folic acid (FA)-functionalized nanogels (FA@Nanogels/DOX) demonstrated higher tumor cell proliferation inhibition effect in B16F10 cancer cells than that in CHO-K1 normal cells, while the non-FA-functionalized counterparts (Nanogels/DOX) do not show similar effect. This work provides a versatile “one-pot RPP-photo Click functionalization” strategy to construct surface-functionalized pH/redox dual responsive nanogels for efficient and targeted drug delivery.     

Assessment of Betulinic Acid Cytotoxicity and Mitochondrial Metabolism Impairment in a Human Melanoma Cell Line

Melanoma represents one of the most aggressive and drug resistant skin cancers with poor prognosis in its advanced stages. Despite the increasing number of targeted therapies, novel approaches are needed to counteract both therapeutic resistance and the side effects of classic therapy. Betulinic acid (BA) is a bioactive phytocompound that has been reported to induce apoptosis in several types of cancers including melanomas; however, its effects on mitochondrial bioenergetics are less investigated. The present study performed in A375 human melanoma cells was aimed to characterize the effects of BA on mitochondrial bioenergetics and cellular behavior. BA demonstrated a dose-dependent inhibitory effect in both mitochondrial respiration and glycolysis in A375 melanoma cells and at sub-toxic concentrations (10 μM) induced mitochondrial dysfunction by eliciting a decrease in the mitochondrial membrane potential and changes in mitochondria morphology and localization. In addition, BA triggered a dose-dependent cytotoxic effect characterized by apoptotic features: morphological alterations (nuclear fragmentation, apoptotic bodies) and the upregulation of pro-apoptotic markers mRNA expression (Bax, Bad and Bak). BA represents a viable therapeutic option via a complex modulatory effect on mitochondrial metabolism that might be useful in advanced melanoma or as reliable strategy to counteract resistance to standard therapy.     

Mechanism and determinants of nanoparticle penetration through human skin

The ability of nanoparticles to penetrate the stratum corneum was the focus of several studies. Yet, there are controversial issues available for particle penetration due to different experimental setups. Meanwhile, there is little known about the mechanism and determinants of their penetration. In this paper the penetration of four model gold nanoparticles of diameter 6 and 15 nm, differing in surface polarity and the nature of the vehicle, through human skin was studied using multiphoton microscopy. This is in an attempt to profoundly investigate the parameters governing particle penetration through human skin. Our results imply that nanoparticles at this size range permeate the stratum corneum in a similar manner to drug molecules, mainly through the intercellular pathways. However, due to their particulate nature, permeation is also dependent on the complex microstructure of the stratum corneum with its tortuous aqueous and lipidic channels, as shown from our experiments performed using skin of different grades of barrier integrity. The vehicle (toluene-versus-water) had a minimal effect on skin penetration of gold nanoparticles. Other considerations in setting up a penetration experiment for nanoparticles were also studied. The results obtained are important for designing a new transdermal carrier and for a basic understanding of skin-nanoparticle interaction.     

Hyperthermia Enhances Doxorubicin Therapeutic Efficacy against A375 and MNT-1 Melanoma Cells

Melanoma is the deadliest form of skin cancer, and its incidence has alarmingly increased in the last few decades, creating a need for novel treatment approaches. Thus, we evaluated the combinatorial effect of doxorubicin (DOX) and hyperthermia on A375 and MNT-1 human melanoma cell lines. Cells were treated with DOX for 24, 48, and 72 h and their viabilities were assessed. The effect of DOX IC10 and IC20 (combined at 43 °C for 30, 60, and 120 min) on cell viability was further analyzed. Interference on cell cycle dynamics, reactive oxygen species (ROS) production, and apoptosis upon treatment (with 30 min at 43 °C and DOX at the IC20 for 48 h) were analyzed by flow cytometry. Combined treatment significantly decreased cell viability, but not in all tested conditions, suggesting that the effect depends on the drug concentration and heat treatment duration. Combined treatment also mediated a G2/M phase arrest in both cell lines, as well as increasing ROS levels. Additionally, it induced early apoptosis in MNT-1 cells, while in A375 cells this effect was similar to the one caused by hyperthermia alone. These findings demonstrate that hyperthermia enhances DOX effect through cell cycle arrest, oxidative stress, and apoptotic cell death.     

One‐step formation of polyurea gel as a multifunctional approach for biological and environmental applications

This work presents a facile one‐pot fabrication of polyurea (PU) organogels based on polyetheramine (PEO, Jeffamine ED‐2003) and a crosslinker hexamethylene diisocyanate trimer (HDI) which were subsequently used as a drug carrier to incorporate a curcumin hydrophobic molecule in aqueous conditions and for removal of an anionic pollutant in water. PU can be obtained in different shapes with easy control of film thickness with high transparency, flexibility and homogeneous dispersion of curcumin in the matrix. Due to the hydrophilic nature of PEO a high swelling degree was demonstrated by the PU gel. The phase separated domains and the temperature's influence on the system's structural order/disorder were examined by small‐angle X‐ray scattering, showing a more PU ordered system (interface between hard and soft segments) after decreasing the temperature to ?90 °C. The presence of curcumin in the structure of PU was confirmed by Fourier transform infrared, photoluminescence and release studies. A highly efficient removal of Congo red dye from aqueous solution using PU as adsorbent was observed. Moreover, the effect of unloaded and curcumin‐loaded PU gels was evaluated on Schistosoma mansoni adult worms. Detailed in vitro cell viability experiments showed the biocompatibility of loaded and unloaded PU gels. The easy processability of the multifunctional PU shows that it has promising applications as transdermal and soft tissue implantable delivery devices for a large number of poorly water‐soluble drugs and as an efficient adsorbent for removal of organic dyes; it can also be used in the future in dermatological treatments against malignant melanoma. © 2020 Society of Chemical Industry