Enhancing plant biotechnology by nanoparticle delivery of nucleic acids.

Plant biotechnology plays a crucial role in developing modern agriculture and plant science research. However, the delivery of exogenous genetic material into plants has been a long-standing obstacle. Nanoparticle-based delivery systems are being established to address this limitation and are proving to be a feasible, versatile, and efficient approach to facilitate the internalization of functional RNA and DNA by plants. The nanoparticle-based delivery systems can also be designed for subcellular delivery and controlled release of the biomolecular cargo. In this review, we provide a concise overview of the recent advances in nanocarriers for the delivery of biomolecules into plants, with a specific focus on applications to enhance RNA interference, foreign gene transfer, and genome editing in plants.

Nanoparticle elasticity regulates the formation of cell membrane-coated nanoparticles and their nano-bio interactions.

Cell membrane-coated nanoparticles are emerging as a new type of promising nanomaterials for immune evasion and targeted delivery. An underlying premise is that the unique biological functions of natural cell membranes can be conferred on the inherent physiochemical properties of nanoparticles by coating them with a cell membrane. However, the extent to which the membrane protein properties are preserved on these nanoparticles and the consequent bio-nano interactions are largely unexplored. Here, we synthesized two mesenchymal stem cell (MSC) membrane-coated silica nanoparticles (MCSNs), which have similar sizes but distinctly different stiffness values (MPa and GPa). Unexpectedly, a much lower macrophage uptake, but much higher cancer cell uptake, was found with the soft MCSNs compared with the stiff MCSNs. Intriguingly, we discovered that the soft MCSNs enabled the forming of a more protein-rich membrane coating and that coating had a high content of the MSC chemokine CXCR4 and MSC surface marker CD90. This led to the soft MCSNs enhancing cancer cell uptake mediated by the CD90/integrin receptor-mediated pathway and CXCR4/SDF-1 pathways. These findings provide a major step forward in our fundamental understanding of how the combination of nanoparticle elasticity and membrane coating may be used to facilitate bio-nano interactions and pave the way forward in the development of more effective cancer nanomedicines.

Advances in Nanomaterials for Immunotherapeutic Improvement of Cancer Chemotherapy.

Immuno-stimulative effect of chemotherapy (ISECT) is recognized as a potential alternative to conventional immunotherapies, however, the clinical application is constrained by its inefficiency. Metronomic chemotherapy, though designed to overcome these limitations, offers inconsistent results, with effectiveness varying based on cancer types, stages, and patient-specific factors. In parallel, a wealth of preclinical nanomaterials holds considerable promise for ISECT improvement by modulating the cancer-immunity cycle. In the area of biomedical nanomaterials, current literature reviews mainly concentrate on a specific category of nanomaterials and nanotechnological perspectives, while two essential issues are still lacking, i.e., a comprehensive analysis addressing the causes for ISECT inefficiency and a thorough summary elaborating the nanomaterials for ISECT improvement. This review thus aims to fill these gaps and catalyze further development in this field. For the first time, this review comprehensively discusses the causes of ISECT inefficiency. It then meticulously categorizes six types of nanomaterials for improving ISECT. Subsequently, practical strategies are further proposed for addressing inefficient ISECT, along with a detailed discussion on exemplary nanomedicines. Finally, this review provides insights into the challenges and perspectives for improving chemo-immunotherapy by innovations in nanomaterials.

A New Sono-Chemo Sensitizer Overcoming Tumor Hypoxia for Augmented Sono/Chemo-Dynamic Therapy and Robust Immune-Activating Response.

Novel sonosensitizers with intrinsic characteristics for tumor diagnosis, efficient therapy, and tumor microenvironment regulation are appealing in current sonodynamic therapy. Herein, a manganese (Mn)-layered double hydroxide-based defect-rich nanoplatform is presented as a new type of sono-chemo sensitizer, which allows ultrasound to efficiently trigger reactive oxygen species generation for enhanced sono/chemo-dynamic therapy. Moreover, such a nanoplatform is able to relieve tumor hypoxia and achieve augmented singlet oxygen production via catalyzing endogenous H2 O2 into O2 . On top of these actions, the released Mn2+ ions and immune-modulating agent significantly intensify immune activation and reverse the immunosuppressive tumor microenvironment to the immunocompetent one. Consequently, this nanoplatform exhibits excellent anti-tumor efficacy and effectively suppresses both primary and distant tumor growth, demonstrating a new strategy to functionalize nanoparticles as sono-chemo sensitizers for synergistic combination cancer therapy.

Clay nanoparticles efficiently deliver small interfering RNA to intact plant leaf cells.

RNA interference is triggered in plants by the exogenous application of double-stranded RNA or small interfering RNA (siRNA) to silence the expression of target genes. This approach can potentially provide insights into metabolic pathways and gene function and afford plant protection against viruses and other plant pathogens. However, the effective delivery of biomolecules such as siRNA into plant cells is difficult because of the unique barrier imposed by the plant cell wall. Here, we demonstrate that 40-nm layered double hydroxide (LDH) nanoparticles are rapidly taken up by intact Nicotiana benthamiana leaf cells and by chloroplasts, following their application via infiltration. We also describe the distribution of infiltrated LDH nanoparticles in leaves and demonstrate their translocation through the apoplast and vasculature system. Furthermore, we show that 40-nm LDH nanoparticles can greatly enhance the internalization of nucleic acids by N. benthamiana leaf cells to facilitate siRNA-mediated downregulation of targeted transgene mRNA by >70% within 1 day of exogenous application. Together, our results show that 40-nm LDH nanoparticle is an effective platform for delivery of siRNA into intact plant leaf cells.

Sheet-like clay nanoparticles deliver RNA into developing pollen to efficiently silence a target gene.

Topical application of double-stranded RNA (dsRNA) can induce RNA interference (RNAi) and modify traits in plants without genetic modification. However, delivering dsRNA into plant cells remains challenging. Using developing tomato (Solanum lycopersicum) pollen as a model plant cell system, we demonstrate that layered double hydroxide (LDH) nanoparticles up to 50 nm in diameter are readily internalized, particularly by early bicellular pollen, in both energy-dependent and energy-independent manners and without physical or chemical aids. More importantly, these LDH nanoparticles efficiently deliver dsRNA into tomato pollen within 2-4 h of incubation, resulting in an 89% decrease in transgene reporter mRNA levels in early bicellular pollen 3-d post-treatment, compared with a 37% decrease induced by the same dose of naked dsRNA. The target gene silencing is dependent on the LDH particle size, the dsRNA dose, the LDH-dsRNA complexing ratio, and the treatment time. Our findings indicate that LDH nanoparticles are an effective nonviral vector for the effective delivery of dsRNA and other biomolecules into plant cells.

Strategy for Cytoplasmic Delivery Using Inorganic Particles.

Endosome escape is a key process for intracellular uptake of intact biomolecules and therapeutics, such as nucleic acids. Lysosome escape is a more common pathway during endocytosis, while some biomolecular, organic and inorganic materials are found to enhance the endosome escape, and several mechanisms have been proposed accordingly. Specifically, some inorganic nanomaterials show their unique mechanisms of action for enhanced endosome escape, including salt osmotic effect and gas blast effect. These inorganic nanomaterials are basically weakly alkaline and are naturally featured with the anti-acidification capacity, with limited solubility in neutral solutions. This review paper has briefly presented the strategies in the design of inorganic nanoparticle-based cellular delivery vehicles with endosome escapability and discussed a few typical inorganic nanomaterials that are currently widely examined for delivery purpose. A brief summary and prospect for this kind of inorganic nanomaterials are provided.

Targeted Molecular Imaging of Cardiovascular Diseases by Iron Oxide Nanoparticles.

Cardiovascular disease is one of the major contributors to global disease burden. Atherosclerosis is an inflammatory process that involves the accumulation of lipids and fibrous elements in the large arteries, forming an atherosclerotic plaque. Rupture of unstable plaques leads to thrombosis that triggers life-threatening complications such as myocardial infarction. Current diagnostic methods are invasive as they require insertion of a catheter into the coronary artery. Molecular imaging techniques, such as magnetic resonance imaging, have been developed to image atherosclerotic plaques and thrombosis due to its high spatial resolution and safety. The sensitivity of magnetic resonance imaging can be improved with contrast agents, such as iron oxide nanoparticles. This review presents the most recent advances in atherosclerosis, thrombosis, and myocardial infarction molecular imaging using iron oxide-based nanoparticles. While some studies have shown their effectiveness, many are yet to undertake comprehensive testing of biocompatibility. There are still potential hazards to address and complications to diagnosis, therefore strategies for overcoming these challenges are required.

BioClay™ prolongs RNA interference-mediated crop protection against Botrytis cinerea.

One of the most promising tools for the control of fungal plant diseases is spray-induced gene silencing (SIGS). In SIGS, small interfering RNA (siRNA) or double-stranded RNA (dsRNA) targeting essential or virulence-related pathogen genes are exogenously applied to plants and postharvest products to trigger RNA interference (RNAi) of the targeted genes, inhibiting fungal growth and disease. However, SIGS is limited by the unstable nature of RNA under environmental conditions. The use of layered double hydroxide or clay particles as carriers to deliver biologically active dsRNA, a formulation termed BioClay™, can enhance RNA durability on plants, prolonging its activity against pathogens. Here, we demonstrate that dsRNA delivered as BioClay can prolong protection against Botrytis cinerea, a major plant fungal pathogen, on tomato leaves and fruit and on mature chickpea plants. BioClay increased the protection window from 1 to 3 weeks on tomato leaves and from 5 to 10 days on tomato fruits, when compared with naked dsRNA. In flowering chickpea plants, BioClay provided prolonged protection for up to 4 weeks, covering the critical period of poding, whereas naked dsRNA provided limited protection. This research represents a major step forward for the adoption of SIGS as an eco-friendly alternative to traditional fungicides.

Biomimetic 2D layered double hydroxide nanocomposites for hyperthermia-facilitated homologous targeting cancer photo-chemotherapy.

BACKGROUND: Multi-modal therapy has attracted increasing attention as it provides enhanced effectiveness and potential stimulation of the immune community. However, low accumulation at the tumor sites and quick immune clearance of the anti-tumor agents are still insurmountable challenges. Hypothetically, cancer cell membrane (CCM) can homologously target the tumor whereas multi-modal therapy can complement the disadvantages of singular therapies. Meanwhile, moderate hyperthermia induced by photothermal therapy can boost the cellular uptake of therapeutic agents by cancer cells. RESULTS: CCM-cloaked indocyanine green (ICG)-incorporated and abraxane (PTX-BSA)-loaded layered double hydroxide (LDH) nanosheets (LIPC NSs) were fabricated for target efficient photo-chemotherapy of colorectal carcinoma (CRC). The CCM-cloaked LDH delivery system showed efficient homologous targeting and cytotoxicity, which was further enhanced under laser irradiation to synergize CRC apoptosis. On the other hand, CCM-cloaking remarkably reduced the uptake of LDH NSs by HEK 293T cells and macrophages, implying mitigation of the side effects and the immune clearance, respectively. In vivo data further exhibited that LIPC NSs enhanced the drug accumulation in tumor tissues and significantly retarded tumor progression under laser irradiation at very low therapeutic doses (1.2 and 0.6 mg/kg of ICG and PTX-BSA), without observed side effects on other organs. CONCLUSIONS: This research has demonstrated that targeting delivery efficiency and immune-escaping ability of LIPC NSs are tremendously enhanced by CCM cloaking for efficient tumor accumulation and in situ generated hyperthermia boosts the uptake of LIPC NSs by cancer cells, a potential effective way to improve the multi-modal cancer therapy.

Albumin-stabilized layered double hydroxide nanoparticles synergized combination chemotherapy for colorectal cancer treatment.

Combination chemotherapy with two or more complimentary drugs has been widely used for clinical cancer treatment. However, the efficacy and side effects of combination chemotherapy still remain a challenge. Here, we constructed an albumin-stabilized layered double hydroxide nanoparticle (BLDH) system to simultaneously load and deliver two widely used anti-tumor drugs, i.e. 5-fluorouracil (5FU) and albumin-bound PTX (Abraxane, ABX) for colorectal cancer treatment. The cellular uptake test has revealed that 5FU-ABX encapsulated BLDH (BLDH/5FU-ABX) nanoparticles were efficiently internalized by the colorectal cancer cell (HCT-116), synergistically inducing apoptosis of colon cancer cells. The in vivo test has demonstrated that BLDH/5FU-ABX nanomedicine significantly inhibited the tumor growth after three intravenous injections, without any detectable side effects. The enhanced therapeutic effectiveness is attributed to efficient accumulation of BLDH/5FU-ABX at tumor sites and acid-sensitive release of co-loaded drugs. Thus, combination chemotherapy based on BLDH/5FU-ABX nanomedicine would be a new strategy for colorectal cancer treatment.

Charge Reversion Simultaneously Enhances Tumor Accumulation and Cell Uptake of Layered Double Hydroxide Nanohybrids for Effective Imaging and Therapy.

Nanotheranostics have been actively sought in precision nanomedicine in recent years. However, insufficient tumor accumulation and limited cell uptake often impede the nanotheranostic efficacy. Herein, pH-sensitive charge-reversible polymer-coated layered double hydroxide (LDH) nanohybrids are devised to possess long circulation in blood but reserve surface charges in the weakly acidic tumor tissue to re-expose therapeutic LDH nanoparticles for enhanced tumor accumulation and cell uptake. In vitro experimental data demonstrate that charge-reversible nanohybrids mitigate the cell uptake in physiological conditions (pH 7.4), but remarkably facilitate internalization by tumor cells after charge reversion in the weakly acidic environment (pH 6.8). More significantly, about 6.0% of injected charge-reversible nanohybrids accumulate in the tumor tissue at 24 h post injection, far higher than the average accumulation (0.7%) reported elsewhere for nanoparticles. This high tumor accumulation clearly shows the tumor tissues in T1 -weighted magnetic resonance imaging. As a consequence, >95% inhibition of tumor growth in the B16F0-bearing mouse model is achieved via only one treatment combining RNAi and photothermal therapy under very mild irradiation (808 nm laser, 0.3 W cm-2 for 180 s). The current research thus demonstrates a new strategy to functionalize nanoparticles and simultaneously enhance their tumor accumulation and cell internalization for effective cancer theranostics.

"Dual-Key-and-Lock" Ruthenium Complex Probe for Lysosomal Formaldehyde in Cancer Cells and Tumors.

Biomedical investigations reveal that excessive formaldehyde generation is possibly a critical factor for tissue cancerization, cancer progression, and metastasis. Responsive molecular probes that can detect lysosomal formaldehyde in live cells and tumors and monitor drug-triggered formaldehyde scavenging contribute potentially to future cancer diagnosis and treatment monitoring. Herein, a novel "dual-key-and-lock" strategy-based ruthenium(II) complex probe, Ru-FA, is reported as an effective tool for formaldehyde detection in vitro and in vivo. Ru-FA shows weak luminescence due to photon-induced electron transfer (PET) process from Ru(II) center to electron withdrawing group 2,4-dinitrobenzene (DNB). Triggered by the specific reaction with formaldehyde (first "key") in an acidic microenvironment (second "key"), DNB is cleaved from Ru-FA, affording an emissive Ru(II) complex derivative, Ru-NR. Spectrometric analysis including steady-state and time-gated luminescence indicates that Ru-FA is favorable to be used as the probe for quantification of formaldehyde in human sera and mouse organs. Ru-FA is biocompatible and cell membrane permeable. Together with its smart "dual-key-and-lock" response to formaldehyde, luminescence imaging of lysosomal formaldehyde in live cells, visualization of tumor-derived endogenous formaldehyde, and monitoring of formaldehyde scavenging in mice were achieved, followed by the successful demonstration on detection of formaldehyde in tumors and other organs. These in vivo and in vitro detection confirm not only the excessive formaldehyde generation in tumors, but also the efficient drug administration to scavenge formaldehyde, demonstrating the potential application of Ru-FA in cancer diagnosis and treatment monitoring through lysosomal formaldehyde detection.

Nanoparticle-Based Nanomedicines to Promote Cancer Immunotherapy: Recent Advances and Future Directions.

Cancer immunotherapy is a promising cancer terminator by directing the patient's own immune system in the fight against this challenging disorder. Despite the monumental therapeutic potential of several immunotherapy strategies in clinical applications, the efficacious responses of a wide range of immunotherapeutic agents are limited in virtue of their inadequate accumulation in the tumor tissue and fatal side effects. In the last decades, increasing evidences disclose that nanotechnology acts as an appealing solution to address these technical barriers via conferring rational physicochemical properties to nanomaterials. In this Review, an imperative emphasis will be drawn from the current understanding of the effect of a nanosystem's structure characteristics (e.g., size, shape, surface charge, elasticity) and its chemical modification on its transport and biodistribution behavior. Subsequently, rapid-moving advances of nanoparticle-based cancer immunotherapies are summarized from traditional vaccine strategies to recent novel approaches, including delivery of immunotherapeutics (such as whole cancer cell vaccines, immune checkpoint blockade, and immunogenic cell death) and engineered immune cells, to regulate tumor microenvironment and activate cellular immunity. The future prospects may involve in the rational combination of a few immunotherapies for more efficient cancer inhibition and elimination.

Integrating Fluorinated Polymer and Manganese-Layered Double Hydroxide Nanoparticles as pH-activated 19 F MRI Agents for Specific and Sensitive Detection of Breast Cancer.

19 F magnetic resonance imaging (19 F MRI) agents capable of being activated upon interactions with cancer triggers are attracting increasing attention, although challenges still remain for precise and specific detection of cancer tissues. In this study, a novel hybrid 19 F MRI agent for pH-sensitive detection of breast cancer tissues is reported, a composite system designed by conjugating a perfluoropolyether onto the surface of manganese-incorporated layered double hydroxide (Mn-LDH@PFPE) nanoparticles. The 19 F NMR/MRI signals from aqueous solutions of Mn-LDH@PFPE nanoparticles are quenched at pH 7.4, but "turned on" following a reduction in pH to below 6.5. This is due to partial dissolution of Mn2+ from the Mn-LDH nanoparticles and subsequent reduction in the effect of paramagnetic relaxation. Significantly, in vivo experiments reveal that an intense 19 F MR signal can be detected only in the breast tumor tissue after intravenous injection of Mn-LDH@PFPE nanoparticles due to such a specific activation. Thus pH-activated Mn-LDH@PFPE nanoparticles are a potential "smart" 19 F MRI agent for precise and specific detection of cancer diseases.

Responsive Upconversion Nanoprobe for Background-Free Hypochlorous Acid Detection and Bioimaging.

Responsive nanoprobes play an important role in bioassay and bioimaging, early diagnosis of diseases and treatment monitoring. Herein, a upconversional nanoparticle (UCNP)-based nanoprobe, Ru@UCNPs, for specific sensing and imaging of hypochlorous acid (HOCl) is reported. This Ru@UCNP nanoprobe consists of two functional components,, i.e., NaYF4 :Yb, Tm UCNPs that can convert near infrared light-to-visible light as the energy donor, and a HOCl-responsive ruthenium(II) complex [Ru(bpy)2 (DNCH-bpy)](PF6 )2 (Ru-DNPH) as the energy acceptor and also the upconversion luminescence (UCL) quencher. Within this luminescence resonance energy transfer nanoprobe system, the UCL OFF-ON emission is triggered specifically by HOCl. This triggering reaction enables the detection of HOCl in aqueous solution and biological systems. As an example of applications, the Ru@UCNPs nanoprobe is loaded onto test papers for semiquantitative HOCl detection without any interference from the background fluorescence. The application of Ru@UCNPs for background-free detection and visualization of HOCl in cells and mice is successfully demonstrated. This research has thus shown that Ru@UCNPs is a selective HOCl-responsive nanoprobe, providing a new way to detect HOCl and a new strategy to develop novel nanoprobes for in situ detection of various biomarkers in cells and early disgnosis of animal diseases.

Iridium(III) Complex-Based Activatable Probe for Phosphorescent/Time-Gated Luminescent Sensing and Imaging of Cysteine in Mitochondria of Live Cells and Animals.

This study reports an activatable iridium(III) complex probe for phosphorescence/time-gated luminescence detection of cysteine (Cys) in vitro and in vivo. The probe, [Ir(ppy)2 (NTY-bpy)](PF6 ) [ppy: 2-phenylpyridine; NTY-bpy: 4-methyl-4'-(2-nitrovinyl)-2,2'-bipyridine], is developed by incorporating a strong electron-withdrawing group, nitroolefin, into a bipyridine ligand of the IrIII complex. The luminescence of the probe is quenched owing to the intramolecular charge transfer (ICT) process, but switched on by a specific recognition reaction between the probe and Cys. [Ir(ppy)2 (NTY-bpy)](PF6 ) shows high sensitivity and selectivity for Cys detection and good biocompatibility. The long-lived emission of [Ir(ppy)2 (NTY-bpy)](PF6 ) allows time-gated luminescence analysis of Cys in cells and human sera. These properties make it convenient for the phosphorescence and time-gated luminescence imaging and flow cytometry analysis of Cys in live samples. The Cys images in cancer cells and inflamed macrophage cells reveal that [Ir(ppy)2 (NTY-bpy)](PF6 ) is distributed in mitochondria after cellular internalization. Visualizations and flow cytometry analysis of mitochondrial Cys levels and Cys-mediated redox activities of live cells are achieved. By using [Ir(ppy)2 (NTY-bpy)](PF6 ) as a probe, in vivo sensing and imaging of Cys in D. magna, zebrafish, and mice are then demonstrated.

Insluin and epithelial growth factor (EGF) promote programmed death ligand 1(PD-L1) production and transport in colon cancer stem cells.

BACKGROUND: Programmed cell death ligand 1 (PD-L1) is an important immune-inhibitory protein expressed on cancer cells to mediate cancer escape through interaction with PD-1 expressed on activated T lymphocytes (T cells). Previously, we reported that colon and breast cancer stem cells (CSCs) expressed much higher levels of PD-L1 than their parental cells, suggesting they will be more resistant to immune attack. METHODS: We investigated the underlining mechanism of PD-L1 increase in colon CSCs, with a special focus on the effect of insulin and epithelial growth factor (EGF), the two fundamental components to sustain the metabolism and stemness in the culture of CSCs. RESULTS: We found that insulin increased the total and surface PD-L1 levels through PI3K/Akt/mTOR pathway as the increase could be inhibited by the dual inhibitor of the pathway, BEZ235. EGF didn't affect the total PD-L1 levels of CSCs but increased the cell surface protein levels by flow cytometry analysis, indicating EGF promotes the transport of PD-L1 to the cell surface. Blocking cell surface PD-L1 with a specific antibody resulted in a significant reduction of tumour sphere formation but didn't interfere with the sphere growth, suggesting that cell surface PD-L1 may act as an adhering molecule for CSCs. CONCLUSIONS: Apart from the essential roles in metabolism and stemness, insulin and EGF involve in up-regulation of PD-L1 expression in colon CSCs, therefore the inhibition of insulin and EGF/EGFR pathways can be considered for cancer immunotherapy or combined with PD-1/PD-L1 antibody-based cancer immunotherapy to eliminate CSCs.

Clay Nanoparticles Elicit Long-Term Immune Responses by Forming Biodegradable Depots for Sustained Antigen Stimulation.

Nanomaterials have been widely tested as new generation vaccine adjuvants, but few evoke efficient immunoreactions. Clay nanoparticles, for example, layered double hydroxide (LDH) and hectorite (HEC) nanoparticles, have shown their potent adjuvanticity in generating effective and durable immune responses. However, the mechanism by which clay nanoadjuvants stimulate the immune system is not well understood. Here, it is demonstrated that LDH and HEC-antigen complexes form loose agglomerates in culture medium/serum. They also form nodules with loose structures in tissue after subcutaneous injection, where they act as a depot for up to 35 d. More importantly, clay nanoparticles actively and continuously recruit immune cells into the depot for up to one month, and stimulate stronger immune responses than FDA-approved adjuvants, Alum and QuilA. Sustained antigen release is also observed in clay nanoparticle depots, with 50-60% antigen released after 35 d. In contrast, Alum-antigen complexes show minimal antigen release from the depot. Importantly, LDH and HEC are more effective than QuilA and Alum in promoting memory T-cell proliferation. These findings suggest that both clay nanoadjuvants can serve as active vaccine platforms for sustained and potent immune responses.

Nano- and micro-materials in the treatment of internal bleeding and uncontrolled hemorrhage.

Internal bleeding is defined as the loss of blood that occurs inside of a body cavity. After a traumatic injury, hemorrhage accounts for over 35% of pre-hospital deaths and 40% of deaths within the first 24 hours. Coagulopathy, a disorder in which the blood is not able to properly form clots, typically develops after traumatic injury and results in a higher rate of mortality. The current methods to treat internal bleeding and coagulopathy are inadequate due to the requirement of extensive medical equipment that is typically not available at the site of injury. To discover a potential route for future research, several current and novel treatment methods have been reviewed and analyzed. The aim of investigating different potential treatment options is to expand available knowledge, while also call attention to the importance of research in the field of treatment for internal bleeding and hemorrhage due to trauma.