Cellular and molecular bioengineering最新文献

Background: Identification and quantitation of newly synthesized proteins (NSPs) are critical to understanding protein dynamics in development and disease. Probing the nascent proteome can be achieved using non-canonical amino acids (ncAAs) to selectively label the NSPs utilizing endogenous translation machinery, which can then be quantitated with mass spectrometry. We have previously demonstrated that labeling the in vivo murine proteome is feasible via injection of azidohomoalanine (Aha), an ncAA and methionine (Met) analog, without the need for Met depletion. Aha labeling can address biological questions wherein temporal protein dynamics are significant. However, accessing this temporal resolution requires a more complete understanding of Aha distribution kinetics in tissues.

Results: To address these gaps, we created a deterministic, compartmental model of the kinetic transport and incorporation of Aha in mice. Model results demonstrate the ability to predict Aha distribution and protein labeling in a variety of tissues and dosing paradigms. To establish the suitability of the method for in vivo studies, we investigated the impact of Aha administration on normal physiology by analyzing plasma and liver metabolomes following various Aha dosing regimens. We show that Aha administration induces minimal metabolic alterations in mice.

Conclusions: Our results demonstrate that we can reproducibly predict protein labeling and that the administration of this analog does not significantly alter in vivo physiology over the course of our experimental study. We expect this model to be a useful tool to guide future experiments utilizing this technique to study proteomic responses to stimuli.

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-023-00760-4.

Introduction: S100A4 promotes the establishment of tumor microenvironment for malignant cancer cells, and knockdown of S100A4 can inhibit tumorigenesis. However, there is no efficient way to target S100A4 in metastatic tumor tissues. Here, we investigated the role of siS100A4-loaded iRGD-modified extracellular vesicles (siS100A4-iRGD-EVs) in postoperative breast cancer metastasis.

Methods: siS100A4-iRGD-EVs nanoparticles were engineered and analyzed using TEM and DLS. siRNA protection, cellular uptake, and cytotoxicity of EV nanoparticles were examined in vitro. Postoperative lung metastasis mouse model was created to investigate the tissue distribution and anti-metastasis roles of nanoparticles in vivo.

Results: siS100A4-iRGD-EVs protected siRNA from RNase degradation, enhanced the cellular uptake and compatibility in vitro. Strikingly, iRGD-modified EVs significantly increased tumor organotropism and siRNA accumulation in lung PMNs compared to siS100A4-EVs in vivo. Moreover, siS100A4-iRGD-EVs treatment remarkedly attenuated lung metastases from breast cancer and increased survival rate of mice through suppressing S100A4 expression in lung.

Conclusions: siS100A4-iRGD-EVs nanoparticles show more potent anti-metastasis effect in postoperative breast cancer metastasis mouse model.

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-022-00757-5.

Introduction: Drug targeting and controlled drug release systems in cancer treatment have many advantages over conventional chemotherapy in terms of limiting systemic toxicity, side effects, and overcoming drug resistance.

Methods and results: In this paper, fabricating nanoscale delivery system composed of magnetic nanoparticles (MNPs) covered with poly-amidoamine (PAMAM) dendrimers and using its advantages were fully used to help the chemotherapeutic drug, Palbociclib, effectively reach tumors, specifically and stay stable in the circulation longer. In order to determine whether conjugate selectivity can be increased for the specific drug type, we have reported different strategies for loading and conjugation of Palbociclib to different generations of magnetic PAMAM dendrimers. The best method leading to the highest amount of Palbociclib conjugation was chosen, and the characterization of the Palbociclib conjugated dendrimeric magnetic nanoparticles (PAL-DcMNPs) were performed. In vitro pharmacological activity of the conjugation was demonstrated by measuring the cell viability and lactate dehydrogenase (LHD) release. Obtained results indicated that PAL-DcMNPs treatment of the breast cancer cell lines, leads to an increase in cell toxicity compared to free Palbociclib. The observed effects were more evident for MCF-7 cells than for MDA-MB231 and SKBR3 cells, considering that viability decreased to 30% at 2.5 µM treatment of PAL-DcMNPs at MCF-7 cells. Finally, in Palbociclib and PAL-DcMNPs treated breast cancer cells, the expression levels of some pro-apoptotic and drug resistance related genes were performed by RT-PCR analysis.

Conclusion: Our knowledge indicates that the proposed approach is novel, and it can provide new insight into the development of Palbociclib targeting delivery system for cancer treatment.

The tumor microenvironment (TME) plays a critical, yet mechanistically elusive role in tumor development and progression, as well as drug resistance. To better understand the pathophysiology of the complex TME, a reductionist approach has been employed to create in vitro microfluidic models called "tumor chips". Herein, we review the fabrication processes, applications, and limitations of the tumor chips currently under development for use in cancer research. Tumor chips afford capabilities for real-time observation, precise control of microenvironment factors (e.g. stromal and cellular components), and application of physiologically relevant fluid shear stresses and perturbations. Applications for tumor chips include drug screening and toxicity testing, assessment of drug delivery modalities, and studies of transport and interactions of immune cells and circulating tumor cells with primary tumor sites. The utility of tumor chips is currently limited by the ability to recapitulate the nuances of tumor physiology, including extracellular matrix composition and stiffness, heterogeneity of cellular components, hypoxic gradients, and inclusion of blood cells and the coagulome in the blood microenvironment. Overcoming these challenges and improving the physiological relevance of in vitro tumor models could provide powerful testing platforms in cancer research and decrease the need for animal and clinical studies.

Introduction: Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease that leads to eventual renal failure. Metformin (MET), an AMP-activated protein kinase (AMPK) activator already approved for type 2 diabetes, is currently investigated for ADPKD treatment. However, despite high tolerability, MET showed varying therapeutic efficacy in preclinical ADPKD studies. Thus, newer strategies have combined MET with other ADPKD small molecule drug candidates, thereby targeting multiple ADPKD-associated signaling pathways to enhance therapeutic outcomes through potential drug synergy. Unfortunately, the off-target side effects caused by these additional drug candidates pose a major hurdle. To address this, our group has previously developed kidney-targeting peptide amphiphile micelles (KMs), which displayed significant kidney accumulation in vivo, for delivering drugs to the site of the disease.

Methods: To mitigate the adverse effects of ADPKD drugs and evaluate their therapeutic potential in combination with MET, herein, we loaded KMs with ADPKD drug candidates including salsalate, octreotide, bardoxolone methyl, rapamycin, tolvaptan, and pioglitazone, and tested their in vitro therapeutic efficacy when combined with free MET. Specifically, after determining the 40% inhibitory concentration for each drug (IC₄₀), the size, morphology, and surface charge of drug-loaded KMs were characterized. Next, drug-loaded KMs were applied in combination with MET to treat renal proximal tubule cells derived from Pkd1flox/-:TSLargeT mice in 2D proliferation and 3D cyst model.

Results: MET combined with all drug-loaded KMs demonstrated significantly enhanced efficacy as compared to free drugs in inhibiting cell proliferation and cyst growth. Notably, synergistic effects were found for MET and KMs loaded with either salsalate or rapamycin as determined by Bliss synergy scores.

Conclusion: Together, we show drug synergy using drug-loaded nanoparticles and free MET for the first time and present a novel nanomedicine-based combinatorial therapeutic approach for ADPKD with enhanced efficacy.

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-022-00753-9.

Introduction: Neurological diseases, including Alzheimer's, Parkinson's diseases, and brain cancers, are reportedly caused by genetic aberration and cellular malfunction. Herbs with bioactive compounds that have anti-oxidant effects such as cordyceps and turmeric, are of interest to clinical applications due to their minimal adverse effects. The aim of study is to develop the nanoencapsulated cordyceps and turmeric extracts and investigate their capability to enhance the biological activity and improve neuronal function.

Methods: Human neuroblastoma SH-SY5Y cells were utilized as a neuronal model to investigate the properties of nanoencapsulated cordyceps or turmeric extracts, called CMP and TEP, respectively. SH-SY5Y cells were treated with either CMP or TEP and examined the biological consequences, including neuronal maturation and neuronal function.

Results: The results showed that both CMP and TEP improved cellular uptake efficiency within 6 h by 2.3 and 2.8 times, respectively. Besides, they were able to inhibit cellular proliferation of SH-SY5Y cells up to 153- and 218-fold changes, and increase the expression of mature neuronal markers (TUJ1, PAX6, and NESTIN). Upon the treatment of CMP and TEP, the expression of dopaminergic-specific genes (LMX1B, FOXA2, EN1, and NURR1), and the secretion level of dopamine were significantly improved up to 3.3-fold and 3.0-fold, respectively, while the expression of Alzheimer genes (PSEN1, PSEN2, and APP), and the secretion of amyloid precursor protein were significantly reduced by 32-fold and 108-fold, respectively. Importantly, the autophagy activity was upregulated by CMP and TEP at 6.3- and 5.5-fold changes, respectively.

Conclusions: This finding suggested that the nanoencapsulated cordyceps and turmeric extracts accelerated neuronal maturation and alleviated neuronal pathology in human neural cells. This paves the way for nanotechnology-driven drug delivery systems that could potentially be used as an alternative medicine in the future for neurological diseases.

Introduction: Obesity is associated with increased breast cancer incidence, recurrence, and mortality. Adipocytes and adipose-derived stem cells (ASCs), two resident cell types in adipose tissue, accelerate the early stages of breast cancer progression. It remains unclear whether obesity plays a role in the subsequent escape of malignant breast cancer cells into the local circulation.

Methods: We engineered models of human breast tumors with adipose stroma that exhibited different obesity-specific alterations. We used these models to assess the invasion and escape of breast cancer cells into an empty, blind-ended cavity (as a mimic of a lymphatic vessel) for up to sixteen days.

Results: Lean and obese donor-derived adipose stroma hastened escape to similar extents. Moreover, a hypertrophic adipose stroma did not affect the rate of adipose-induced escape. When admixed directly into the model tumors, lean and obese donor-derived ASCs hastened escape similarly.

Conclusions: This study demonstrates that the presence of adipose cells, independently of the obesity status of the adipose tissue donor, hastens the escape of human breast cancer cells in multiple models of obesity-associated breast cancer.

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-022-00750-y.

Introduction: Metabolic strategies in different microenvironments can affect cancer metabolic adaptation, ultimately influencing the therapeutic response. Understanding the metabolic alterations of cancer cells in different microenvironments is critical for therapeutic success.

Methods: In this study, we cultured non-small cell lung cancer cells in three different microenvironments (two-dimensional (2D) plates, soft elastic three-dimensional (3D) porous 2 wt% scaffolds, and stiff elastic 3D porous 4 wt% scaffolds) to investigate the effects of different matrix elasticity as well as 2D and 3D culture settings on the metabolic adaptation of cancer cells.

Results: The results revealed that PGC-1α expression is sensitive to the elasticity of the 3D scaffold. PGC-1α expression was markedly increased in cancer cells cultured in stiff elastic 3D porous 4 wt% scaffolds compared with cells cultured in soft elastic 3D porous 2 wt% scaffolds or 2D plates, enhancing mitochondrial biogenesis and oxidative stress resistance of non-small cell lung cancer through increased reactive oxygen species (ROS) detoxification capacity. However, phosphofructokinase-1 (PFK-1) expression, a key rate-limiting enzyme in glycolysis, did not change significantly in the three microenvironments, indicating that microenvironments may not affect the early stage of glycolysis. Conversely, monocarboxylate transporter 1 (MCT1) expression in 3D culture was significantly reduced compared to 2D culture but without significant difference between soft and stiff scaffolds, indicating that MCT1 expression is more sensitive to the shape of the different cultures of 2D and 3D microenvironment surrounding cells but is unaffected by the scaffold elasticity.

Conclusions: Together, these results demonstrate that differences in the microenvironment of cancer cells profoundly impact their metabolic response.