Quantitative Assessment of Nanoparticle Biodistribution by Fluorescence Imaging, Revisited

Fluorescence-based whole-body imaging is widely used in the evaluation of nanoparticles (NPs) in small animals, often combined with quantitative analysis to indicate their spatiotemporal distribution following systemic administration. An underlying assumption is that the fluorescence label represents NPs and the intensity increases with the amount of NPs and/or the labeling dyes accumulated in the region of interest. We prepare DiR-loaded poly­(lactic-<i>co</i>-glycolic acid) (PLGA) NPs with different surface layers (polyethylene glycol with and without folate terminus) and compare the distribution of fluorescence signals in a mouse model of folate-receptor-expressing tumors by near-infrared fluorescence whole-body imaging. Unexpectedly, we observe that fluorescence distribution patterns differ far more dramatically with DiR loading than with the surface ligand, reaching opposite conclusions with the same type of NPs (tumor-specific delivery <i>vs</i> predominant liver accumulation). Analysis of DiR-loaded PLGA NPs reveals that fluorescence quenching, dequenching, and signal saturation, which occur with the increasing dye content and local NP concentration, are responsible for the conflicting interpretations. This study highlights the critical need for validating fluorescence labeling of NPs in the quantitative analysis of whole-body imaging. In light of our observation, we make suggestions for future whole-body fluorescence imaging in the <i>in vivo</i> evaluation of NP behaviors

Propylene Glycol-Linked Amino Acid/Dipeptide Diester Prodrugs of Oleanolic Acid for PepT1-Mediated Transport: Synthesis, Intestinal Permeability, and Pharmacokinetics

In our previous studies, ethylene glycol-linked amino acid diester prodrugs of oleanolic acid (OA), a Biopharmaceutics Classification System (BCS) class IV drug, designed to target peptide transporter 1 (PepT1) have been synthesized and evaluated. Unlike ethylene glycol, propylene glycol is of very low toxicity in vivo. In this study, propylene glycol was used as a linker to further compare the effect of the type of linker on the stability, permeability, affinity, and bioavailability of the prodrugs of OA. Seven diester prodrugs with amino acid/dipeptide promoieties containing l-Val ester (<b>7a</b>), l-Phe ester (<b>7b</b>), l-Ile ester (<b>7c</b>), d-Val-l-Val ester (<b>9a</b>), l-Val-l-Val ester (<b>9b</b>), l-Ala-l-Val ester (<b>9c</b>), and l-Ala-l-Ile ester (<b>9d</b>) were designed and successfully synthesized. In situ rat single-pass intestinal perfusion (SPIP) model was performed to screen the effective permeability (<i>P</i>_eff) of the prodrugs. <i>P</i>_eff of <b>7a</b>, <b>7b</b>, <b>7c</b>, <b>9a</b>, <b>9b</b>, <b>9c</b>, and <b>9d</b> (6.7-fold, 2.4-fold, 1.24-fold, 1.22-fold, 4.15-fold, 2.2-fold, and 1.4-fold, respectively) in 2-(<i>N</i>-morpholino)­ethanesulfonic acid buffer (MES) with pH 6.0 showed significant increase compared to that of OA (<i>p</i> < 0.01). In hydroxyethyl piperazine ethanesulfonic acid buffer (HEPES) of pH 7.4, except for <b>7c</b>, <b>9a</b>, and <b>9d</b>, <i>P</i>_eff of the other prodrugs containing <b>7a</b> (5.2-fold), <b>7b</b> (2.0-fold), <b>9b</b> (3.1-fold), and <b>9c</b> (1.7-fold) exhibited significantly higher values than that of OA (<i>p</i> < 0.01). In inhibition studies with glycyl-sarcosine (Gly-Sar, a typical substrate of PepT1), <i>P</i>_eff of <b>7a</b> (5.2-fold), <b>7b</b> (2.0-fold), <b>9b</b> (3.1-fold), and <b>9c</b> (2.3-fold) had significantly reduced values (<i>p</i> < 0.01). Compared to the apparent permeability coefficient (<i>P</i>_app) of OA with Caco-2 cell monolayer, significant enhancement of the <i>P</i>_app of <b>7a</b> (5.27-fold), <b>9b</b> (3.31-fold), <b>9a</b> (2.26-fold), <b>7b</b> (2.10-fold), <b>7c</b> (2.03-fold), <b>9c</b> (1.87-fold), and <b>9d</b> (1.39-fold) was also observed (<i>p</i> < 0.01). Inhibition studies with Gly-Sar (1 mM) showed that <i>P</i>_app of <b>7a</b>, <b>9b</b>, and <b>9c</b> significantly reduced by 1.3-fold, 1.6-fold, and 1.4-fold (<i>p</i> < 0.01), respectively. These results may be attributed to PepT1-mediated transport and their differential affinity toward PepT1. According to the permeability and affinity, <b>7a</b> and <b>9b</b> were selected in the pharmacokinetic studies in rats. Compared with group OA, <i>C</i>_max for group <b>7a</b> and <b>9b</b> was enhanced to 3.04-fold (<i>p</i> < 0.01) and 2.62-fold (<i>p</i> < 0.01), respectively. AUC_0→24 was improved to 3.55-fold (<i>p</i> < 0.01) and 3.39-fold (<i>p</i> < 0.01), respectively. Compared to the ethylene glycol-linked amino acid diester prodrugs of OA in our previous work, results from this study revealed that part of the propylene glycol-linked amino acid/dipeptide diester prodrugs showed better stability, permeability, affinity, and bioavailability. In conclusion, propylene glycol-linked amino acid/dipeptide diester prodrugs of OA may be suitable for PepT1-targeted prodrugs of OA to improve the oral bioavailability of OA

Ethylene Glycol-Linked Amino Acid Diester Prodrugs of Oleanolic Acid for PepT1-Mediated Transport: Synthesis, Intestinal Permeability and Pharmacokinetics

The purposes of this study were to expand the structure of parent drugs selected for peptide transporter 1 (PepT1)-targeted ester prodrug design and to improve oral bioavailability of oleanolic acid (OA), a Biopharmaceutics Classification System (BCS) class IV drug. Through an ethoxy linker the carboxylic acid group of OA was conjugated with the carboxylic acid group of different amino acid promoieties to form six diester prodrugs. The effective permeability (<i>P</i>_eff) of prodrugs was screened by in situ rat single-pass intestinal perfusion (SPIP) model in two buffers with different pH (6.0 and 7.4) as PepT1 employs a proton-gradient as the driving force. Compared to OA, 2.5-fold, 2.3-fold, 2.2-fold, 2.1-fold, and 1.9-fold enhancement of <i>P</i>_eff in buffer with pH 6.0 was observed for l-Phe ester (<b>5c</b>), l-Val ester (<b>5a</b>), l-Lys ester (<b>5e</b>), d-Phe ester (<b>5d</b>), and d-Val ester (<b>5b</b>), respectively. Furthermore, <i>P</i>_eff of <b>5a</b>, <b>5c</b>, <b>5d</b> and <b>5e</b> in pH 6.0 was significantly higher than that in pH 7.4 (<i>p</i> < 0.01), respectively. These results showed that the H⁺ concentration of perfusion solution had great effect on the transport of the prodrugs across intestinal membrane. For the further evaluation of affinity to PepT1, inhibition studies were performed by coperfusing 0.1 mM prodrug with 50 mM glycyl-sarcosine (Gly-Sar, a typical substrate of PepT1). It turned out that the <i>P</i>_eff of <b>5a</b>, <b>5b</b>, <b>5c</b> and l-Tyr ester (<b>6f</b>) significantly reduced in the presence of Gly-Sar (1.7-fold, 2.2-fold, 1.9-fold, and 1.4-fold, respectively). We supposed that it may be attributed to PepT1 mediated transport of these prodrugs. <b>5a</b> and <b>6f</b> were selected as the optimal target prodrugs for oral absorption <i>in vivo</i>. Following intragastric administration of 300 mg/kg (calculated as OA) <b>5a</b>, <b>6f</b> and OA in three groups of rats, compared with group OA, <i>C</i>_max for the group of <b>5a</b> and <b>6f</b> was enhanced by 1.56-fold and 1.54-fold, respectively. <i>F</i>_app of group <b>5a</b> and <b>6f</b> was 2.21- and 2.04-fold increased, respectively, indicating that <b>5a</b> and <b>6f</b> had better oral absorption than OA. The combined results also suggest that diester prodrugs which conjugated two carboxylic acid groups of proper amino acid promoieties and parent drug through a linker can be used for PepT1-targeted prodrug design. With this strategy, oral bioavailability of OA in rats could be improved significantly

Mitoxantrone- and Folate-TPGS2k Conjugate Hybrid Micellar Aggregates To Circumvent Toxicity and Enhance Efficiency for Breast Cancer Therapy

Mitoxantrone (MTO) is a potent drug used to treat breast cancer; however, efforts to expand its clinical applicability have been restricted because of its high risk for cardiotoxicity. In this study, we successfully conjugated MTO or folic acid (FA) to a synthesized D-α-tocopheryl polyethylene glycol 2000 succinate (TPGS2k), herein, shortened to MCT and FCT, respectively. The two produced conjugates could self-assemble to form MCT micelles or MCT/FCT mixed micelles (FMCT) aiming to lower systemic toxicity, enhance entrapment efficiency, and provide a platform for targeted delivery. Moreover, these micellar materials showed a significantly low CMC and could be used to load MTO. The diameters of MTO-loaded micelles (MTO-MCT and MTO-FMCT) were less than 100 nm with a negative zeta potential. We further characterized the pH-responsive drug release of MTO-MCT and MTO-FMCT and then assessed their cellular uptake and antitumor efficacy in human breast cancer cell lines (MCF-7) via confocal microscopy, flow cytometry, and cytotoxicity studies. All the results revealed that both MTO-MCT and MTO-FMCT increased drug loading and entrapment efficiency and possessed sufficient pH-sensitive release. Additionally, MTO-FMCT displayed an improved uptake through folate-mediated endocytosis, resulting in a higher cytotoxic effect on MCF-7 cells compared with that of MTO-MCT. Meanwhile, both MTO-MCT and MTO-FMCT exhibited a low toxicity on hCMEC/D3 normal cells. More importantly, pharmacokinetic study demonstrated that, in comparison with free MTO injection, MTO-MCT and MTO-FMCT, respectively, achieved half-lives 11.5 and 13 times longer and a 9.7- and 5.8-fold increase in AUC. In vivo, both MTO-MCT and MTO-FMCT formulations significantly prolonged the survival time of MCF-7 tumor-bearing mice and had a better efficacy/toxicity ratio. Promisingly, MTO-FMCT micelles remarkably increased MTO accumulation in tumors in vivo, induced higher tumor cell apoptosis, and showed lower toxicity toward major organs. These results imply that MTO-FMCT may be used as a potential drug delivery system for breast cancer targeted therapy

A Collaborative Assembly Strategy for Tumor-Targeted siRNA Delivery

A novel “collaborative assembly” approach was reported for the synthesis of an siRNA delivery system via a combination of an electrostatically driven physical assembly and a facile click reaction-mediated chemical assembly, which showed various advantages of more safety, efficiency, and flexibility over the conventional approach that is only based on the physical assembly. This strategy remained a high cationic property of lipid-based complex for high siRNA loading capacity. The direct chemical modification of a model polyanion, hyaluronic acid (HA) on the cationic complex via click chemistry shielded the positive charge of complex without affecting the siRNA binding, which reduced the toxicity and enhanced the blood stability of the complex. In addition, the incorporated polyanion might be prefunctionalized, which endued the carrier with better biological characteristics such as long circulating or tumor targeting. We demonstrated that the obtained lipid-polymer hybrid nanoparticle (RSC-HA) using collaborative assembly presented greater <i>in vivo</i> stability in the blood for efficient tumor targeting than the physically assembled RSC/HA in which HA was physically adsorbed on the complex. After endocytosis into the cells, the protection of RSC-HA on siRNA turned off, while the release of siRNA induced by the intracellular signals for enhanced gene-silencing capacity. This combination of physical and chemical assemblies provides an efficient strategy for the exploitation of safe, stable, and functionalized siRNA delivery systems

Multistep Targeted Nano Drug Delivery System Aiming at Leukemic Stem Cells and Minimal Residual Disease

Refractory leukemia remains the most common therapeutic problem in clinical treatment of leukemia. The key therapy of refractory leukemia is to kill, thoroughly, the minimal residual disease and leukemia stem cells in the highly vascularized red marrow areas. In this study, two new conjugates, alendronate-polyethylene glycol (100) monostearate and folate-polyethylene glycol (100) monostearate, were synthesized to develop a multistep targeting nanostructured lipid carriers by enhancing drug transport to the high bone turnover areas adjacent to the red marrow and targeting the minimal residual disease and leukemia stem cells. This dual targeting system demonstrated a great binding affinity to hydroxyapatite, a model component of bone minerals, and higher cell uptake (in the form of carriers but not drug) and cytotoxicity in the K562 cell line, a leukemia cell line with overexpressed folate receptors, were observed <i>in vitro</i> compared to unmodified carriers, especially when the cells were pretreated and the receptors were up-regulated by all-<i>trans</i> retinoic acid. The comodel test of K562 cells and HA showed that this dual targeting system could desorb from bone surface and be taken up by leukemia cells. For the <i>in vivo</i> study, this dual targeting system exhibited a significant increase in plasma half-life and could specifically accumulate in the bone tissue of rats or mice after intravenous injection. <i>Ex vivo</i> imaging of mice femurs and confocal laser scanning microscope imaging of mice femur slices further confirmed that this dual targeting system could favorably deposit to the osteoblast-enriched areas of high bone turnover in regions of trabecular bone surrounded by red marrow. <i>In vivo</i> antitumor activity in K562/BALB/c-nu leukemia mice showed that the treatment of this dual targeting system significantly reduced the white blood cell (WBC) number in peripheral blood and bone marrow to the normal level. In conclusion, this dual targeting system could precisely target to the regions where the minimal residual disease and leukemia stem cells are located and then be specifically uptaken in large amounts, which is a valuable target for refractory leukemia therapy

Nanostructured Peptidotoxins as Natural Pro-Oxidants Induced Cancer Cell Death via Amplification of Oxidative Stress

Melittin (Mel), one of the host defense peptides derived from the venom of honeybees, demonstrates substantial anticancer properties, which is attributed to augmenting reactive oxygen species (ROS) generation. However, little has been reported on its pro-oxidation capacity in cancer oxidation therapy. In this study, an ROS amplifying nanodevice was fabricated through direct complexation of two natural pro-oxidants, Mel and condensed epigallocatechin gallate (pEGCG). The obtained nanocomplex (NC) was further covered with phenylboronic acid derivatized hyaluronic acid (pHA) through the ROS-responsive boronate ester coordination bond to produce pHA-NC. Upon undergoing receptor-mediated endocytosis into cancer cells, the inner cores of pHA-NC will be partially uncovered once pHA corona is degraded by hyaluronidase and will then escape from the lysosome by virtue of cytolytic Mel. The elevated ROS level in the tumor cytoplasm can disrupt the boronate ester bond to facilitate drug release. Both Mel and pEGCG could synergistically amplify oxidative stress and prolong ROS retention in cancer cells, leading to enhanced anticancer efficacy. This ROS cascade amplifier based on selective coordination bond and inherent pro-oxidation properties of natural ingredients could detect and elevate intracellular ROS signals, potentiating to move the tumor away from its homeostasis and make the tumor vulnerable. Compared to previously reported chemosynthetic pro-oxidants, the ROS self-sufficient system, fully composed of natural medicine, from this study provides a new insight in developing cancer oxidation therapy