Abstract

Cancer vaccines based on tumor cell components have shown promising results in animal and clinical studies. The vaccine system contains abundant tumor antigen components, which can activate the immune system by antigens. However, their efficacy has been limited by the inability of antigens delivery, which are the core components of vaccines, further fail to be presented and activation of effective cells. Nanotechnology offers a novel platform to enhance the immunogenicity of tumor-associated antigens and deliver them to antigen-presenting cells (APCs) more efficiently. In addition, nanotreatment of tumor cells derivate active ingredients could also help improve the effectiveness of cancer vaccines. In this review, we summarize recent advances in the development of cancer vaccines by the combination of nanotechnology and tumor-based ingredients, including liposomes, polymeric nanoparticles, metallic nanoparticles, virus-like particles and tumor cells membrane, tumor lysate, and specific tumor antigens. These nanovaccines have been designed to increase antigen uptake, prolong antigen presentation, and modulate immune responses through codelivery of immunostimulatory agents. We also further discuss challenges and opportunities in the clinical translation of these nanovaccines.

Introduction

Cancer immunotherapy has revolutionized cancer treatment by harnessing the immune system to recognize and attack cancer cells [1–3]. Among various immunotherapeutic approaches, cancer vaccines have attracted significant attention due to their potential to induce long-lasting and specific immune responses against tumor cells [4–7]. By comparison with surgery, it hardly produce large wounds, nor does it cause higher toxicity in normal tissues and drug tolerance like chemotherapy in the treatment of tumor. However, the clinical efficacy of cancer vaccines based on tumor cell components, such as peptides and proteins, is limited by their poor immunogenicity and inefficient delivery to antigen presentation cells (APCs). Although, many cancer vaccines were applied with the immune-stimulatory agents, such as the programmed cell death protein 1 (PD-1), CpG oligonucleotide, etc. The clinical response rates remain low, especially in the hypoimmunogenic tumors. As we all know, pancreatic cancer is a more difficult type for immunotherapy as a low immunogenic tumor. Therefore, there are few effective cancer vaccines that can actively promote the pancreatic cancer treatment. Meanwhile, limited by the specific tumor antigens, the development of cancer vaccines was extremely difficult.

The field of cancer vaccines has seen significant advancements in recent years, with numerous studies focusing on different aspects of vaccine development and application [8–12]. These advances have been facilitated by the exploration of various types of cancer vaccines, including those targeting viral antigens, neoantigens, and other tumor-specific antigens [13–16]. One approach in cancer vaccine research involves targeting shared antigens from viral infections linked to certain cancer types, such as Epstein–Barr virus and HPV. Studies have shown promising results with vaccines targeting viral antigens like Epstein–Barr nuclear antigen 1 and latent membrane proteins, as well as HPV-related antigens, demonstrating favorable antitumor efficacy and the potential to establish durable T cell memory in rapidly progressing HPV-positive tumors [17]. Another significant area of research is neoantigen cancer vaccines. Neoantigens, derived from nonsynonymous cell variants, are only expressed in tumor cells and can bypass thymus-negative selection, leading to robust neoantigen-specific T cell responses [14]. Advances in genomic and transcriptional profiling have made identifying putative neoantigens possible, although challenges remain in predicting which neoantigens can effectively induce neoantigen-specific T cell responses [18–20]. Cancer vaccines are being developed to stimulate antitumor immunity with tumor antigens delivered in various forms, such as cells, peptides, viruses, and nucleic acids [21–24]. These vaccines aim to overcome tumor heterogeneity and reverse the immunosuppressive microenvironment. Challenges in the field include single antigen targeting, weak immunogenicity, off-target effects, and impaired immune response [25–27]. Current research and reviews provide insights into the principle of action, components, classification, delivery methods, and progress of cancer vaccines, offering a broader perspective for future vaccine design.

Recent studies have shown that tumor antigens can be used for the design of cancer vaccines without the necessity to identify, parse, and reconstruct certain antigens [28–31]. A novel concept has been proposed that the whole antigen vaccine systems derived from tumor cells could be designed to conquer cancer treatments [32–34]. The tumor cells themselves are a huge antigen pool with countless tumor antigen epitopes, which can be used to activate antitumor immune response. Among them, more studies have focused on the application of tumor cell membrane as tumor antigens to stimulate APCs and further activate the antitumor immune response of T cells. In addition, the activation of T cells in immune response requires the involvement of costimulatory molecules. Some studies are based on this theoretical foundation, using genetic engineering to modify tumor cell membranes. By retaining antigen on the cell membrane and inducing the expression of costimulatory molecule CD80, T cells can be directly activated, thereby achieving antitumor immune response [35] (Figure 1). Of course, many studies combine tumor cell membranes that retain tumor antigens with other functional molecules to directly activate dendritic cells (DCs) for antigen presentation, leading to the activation of T cell immune response. These are two different approaches aimed at achieving the same goal [36–38]. A series of nanovaccines developed on this basis have been widely used in the treatment of different tumor cells and preclinical experiments.

Abstract

Introduction

Materials and methods

Characterizations

Results and discussion

Optimized reaction conditions

FTIR spectra analysis

SEM–EDS analysis

XRD analysis

TGA analysis

Conclusion

Declarations

References

Abstract

     An interpenetrating polymer network (IPN) containing gum acacia (GA), poly(methacrylic acid) (MAA), and poly(acrylic acid) (AA) was developed using a two-step aqueous polymerization method. Firstly, semi-IPNs were produced by radical polymerization of MAA chains onto GA in the presence of ammonium persulfate as a free radical initiator and N, N′-methylene-bisacrylamide (MBA) as a cross-linking agent using a microwave heating. To obtain a semi-IPN with a higher swelling percentage, several reaction parameters such as initiator, monomer, and crosslinker concentrations were varied. The percentage swelling (%S) was highly dependent upon the reaction conditions. The optimal reaction conditions for maximal %S were 2.55 × 10–2 mol/L initiator concentration, 12 mL solvent, 0.424 × 10–3 mol/L of monomer, and 2.16 × 10–2 mol/L cross-linker concentration, according to the findings. GA-g-poly(MAA) was the name to given to the semi-IPN. Second, IPN was created by grafting AA chains onto a GA-g-poly(MAA) matrix that had been optimized. The IPN was named as a GA-g-poly(MAA-IPN-AA). The reduction of silver ions to silver nanoparticles (AgNPs) was carried out by heating the mixture of flower extract of Koelreuteria apiculate under microwave radiation.

Introduction

     Hydrogels are three-dimensional (3D) networks formed by the crosslinking of hydrophilic polymers [1, 2]. They might swell while maintaining their network structure by absorbing signifcant amounts of water or biological fluids. The inclusion of hydrophilic groups such as -OH, -CONH, -CONH2, -COOH, and -SO3H along the polymer chain contributes to their ability to absorb a large amount of water [1]. Polysaccharide hydrogel networks have received a lot of attention in recent years [3–5]. The physico-chemical properties of these hydrogels frequently differ significantly from those of the macromolecular constituents. Hydrogel characteristics can also be altered utilizing a range of physical and chemical crosslinking techniques [3, 6]. Various researchers have examined a number of polymeric systems to create hydrogels [7]. The polymer composition can be classified as natural polymeric hydrogels, synthetic polymer hydrogels, and or a combination of both [7]. Because of their non-toxicity, low cost, permeability, and biocompatibility, hydrogels have gotten a lot of attention in a variety of sectors [8–10].

Conclusion

     Antimicrobial materials have been studied extensively in response to rising microbial resistance to traditional antibiotics and disinfectants. Natural polysaccharidebased hydrogels and their composites containing metal nanoparticles could be suitable for antibacterial applications in some cases. Using a microwave irradiation approach, we were able to successfully synthesis GA-gpoly(MAA) and GA-g-poly(MAA-IPN-AA) based hydrogels from GA. APS and MBA being used as initiator and cross-linker, respectively. To achieve maximum %S of semi-IPN (588%) and IPN (500%), diferent parameters were optimized. The formation of cross-linked hydrogels and hydrogel composites with AgNPs was successfully demonstrated using FTIR analysis. The SEM investigation of hydrogel networks reveals a variety of morphologies, which could be attributed to structural diferences between semi-IPN, IPN, and their composites with AgNPs. SemiIPN, IPN and their composite showed superb antibacterial potential against E. coli, M. luteus, P. aeruginosa, Rihzobium species, and S. aureus, and may be used to treat a variety of infected efuents. Such interconnected hydrogels will give superior material properties, particularly in biomedical research, to prevent biomaterialassociated infections as well as general pathogenic invasions into the body. The synthesized hydrogel samples will lead to enhanced applications in the future, as well as a deeper knowledge of material interactions. The eforts to improve the property profle of cross-linked hydrogels are still on, with the hope of improving overall performance in terms of water uptake capacity, biocompatibility, and biodegradability.

Abstract

1. Introduction

2. Materials and Methods

3. Proposed Epilepsy Classification Method

4. Results and Discussion

5. Conclusions

Data Availability

Conflicts of Interest

References

Abstract

     The precise detection of epileptic seizure helps to prevent the serious consequences of seizures. As the electroencephalogram (EEG) reflects the brain activity of patients effectively, it has been widely used in epileptic seizure detection in the past decades. Recently, deep learning-based detection methods which automatically learn features from the EEG signals have attracted much attention. However, with deep learning-based detection methods, different input formats of EEG signals will lead to different detection performances. In this paper, we propose a deep learning-based epileptic seizure detection method with hybrid input formats of EEG signals, i.e., original EEG, Fourier transform of EEG, short-time Fourier transform of EEG, and wavelet transform of EEG. Convolutional neural networks (CNNs) are designed for extracting latent features from these inputs. A feature fusion mechanism is applied to integrate the learned features to generate a more stable syncretic feature for seizure detection. The experimental results show that our proposed hybrid method is effective to improve the seizure detection performance in few-shot scenarios.

Introduction

     Approximately one percent of the world’s population, 65 million people, suffer from epilepsy, more than Parkinson’s disease, Alzheimer’s disease, and multiple sclerosis combined [1]. About two-thirds of people with epilepsy can be treated with medication, and the rest may require surgical intervention. Epilepsy has the characteristics of sudden and recurrent seizures, which may lead to falls, asphyxia, and even death. Therefore, seizure detection is very important for early warning and treatment of epilepsy.

     Epileptic seizure detection is mainly based on electroencephalogram (EEG) [2–4]. Single-channel EEG acquisition equipment improves the practicability of EEG in epileptic detection due to its simplicity in implementation. However, the provided information by signal-channel EEG signal is limited because of the small number of channels. Therefore, it is worth studying to establish a model with high accuracy and high robustness for single-channel EEG epileptic detection.

     The traditional methods are mainly based on feature engineering techniques which extract the corresponding features from EEG signals and then complete the detection based on the extracted features [5–9]. These features include time-domain features [10–12], frequency-domain features [8, 9], and time-frequency-domain features [13–15]. Once the features are extracted, EEG signals can be classified using a variety of classifiers. No matter what classifier is used, the quality of designed features will greatly affect the performance of epilepsy detection. In recent years, with the development of deep learning technology, many works have applied deep learning to perform epilepsy detection [16, 17]. Different from traditional feature engineering, deep learning methods automatically learn features from EEG signals and further complete detection tasks with an end-to-end manner without complicated manual feature design process and can achieve better performance than traditional methods in many scenarios.

Conclusions

     In this paper, we focus on epileptic classification in few-shot scenarios. In order to make the classification accuracy higher and more stable, we propose a deep learning method with hybrid input, i.e., original EEG signal, FFT, STFT, and DWT of EEG signal. In order to alleviate the tendency of overfitting, two means are applied. The first is that we replace the traditional convolution by depthwise separable convolution for reducing the parameters in network and then 2 regularization is applied, whose function is decreasing the complexity of the model. We conduct several experiments to distinguish normal and epileptic EEG, and the results show the proposed method with hybrid input has strong advantages in epileptic classification. It benefits from the complementarity of time-domain properties, frequency-domain properties, and time-frequency-domain properties. Our proposed method provides a new perspective to enrich the input information to make improvements for deep learning-based epileptic diagnosis.

Abstract

     Wastewater from pharmaceutical industries contains different antibiotics and other effluents that directly affect the environment. For the degradation of antibiotics, nanotechnology is an emerging field in research that majorly focuses on molecules and atoms. Nanoparticles are fine micro-particles with more than one dimension from a size of 1–100 nm. The synthesized nanoparticles can directly be used for the process of degradation of antibiotics in wastewater. Titanium dioxide nanoparticles are majorly used among nanoparticles due to their small surface area and particle size. There are various physical, chemical and conventional (biological) methods for synthesizing titanium dioxide nanoparticles. Green synthesis is an environmentally friendly, less expensive and harmless approach, and these methods are much more efficient than any other physical-chemical means. Titanium dioxide nanoparticles are used for different purposes. In food products, these nanoparticles act as photocatalysts and are also used as a coating material in food packaging. Electrochemical biosensors are combined with titanium dioxide nanoparticles for developing nano sheet matrices. Furthermore, this review describes the usage of biological components (plants, microorganisms like fungi and bacteria) to synthesize titanium dioxide nanoparticles. The present review also focuses on applying synthesized titanium dioxide nanoparticles in various drug delivery systems and other bioactivities.

Introduction

     Water is one of the important components of life used in the domestic, industrial, and agricultural sectors. The increase in consumption leads to an increase in the toxicity of pollutants inside water. The concentrations of emerging pollutants like pharmaceutical compounds have increased [1]. Various types of pharmaceutical compounds are observed in the aqueous environment, including antibiotics. These compounds reach the aquatic habitat through hospital effluents, the pharmaceutical industry and human livestock. The consumption of antibiotics has increased, and the concentration increases inside the aquatic environment, leading to the development of antibiotic-resistant bacteria [2,3].

     To remove antibiotics from wastewater, the traditional sewage treatment method is not appropriate and is not much effective in removing the residuals of antibiotics. Nanoparticles can be used as a photocatalyst for the degradation of antibiotics and other organic pollutants [4]. Nanotechnology is defined as synthesizing and developing various nanomaterials by manipulating the matter at the nanoscale [5]. Nanomaterials are a bridge between molecular material and bulk material. They have a size range of 1–100 nm due to their different shapes, sizes, large surface area, and possess high reactivity [6]. This has become an emerging field in science in the last few years due to its wide range of applications [7]. Because of its small size, the effect can be seen in the physical, optical, chemical properties, melting point, and dielectric constant of various nanoparticles [8].

Conclusion

     The interest in green synthesis is due to its advantages of being innocuous to the environment. Different parts of plants are used for synthesis purposes, and their characterization using various techniques helps form TiO2 NPs of varied shapes and sizes. The green synthesis of TiO2 NPs can be used in biomedical, and the delivery of drugs and antibiotics in wastewater of pharmaceutical industries can be degraded using nanoparticles. Because of the less use of precursors, TiO2 is widely used to remove microbes from water surfaces. The nanoparticles inhibit the growth of microbes and are used in wound dressing. Moreover, nanoparticles synthesized using the green routes are more effective than chemical or physical means.

Abstract

1. Introduction

2. Fundamentals of nanotechnology

3. The role of nanotechnology in achieving food security

4. Nanotubes in food and agriculture

5. Nanosensors in food and agriculture

6. The challenges of nanotechnology in food and agriculture system

7. Conclusions

References

Abstract

     Ensuring food security in developing countries is highly challenging due to low productivity of the agriculture sector, degradation of natural resources, high post farming losses, less or no value addition, and high population growth. Researchers are striving to adopt newer technologies to enhance supply to narrow the food demand gap. Nanotechnology is one of the promising technologies that could improve agricultural productivity via nano fertilizers, use of efficient herbicides and pesticides, soil feature regulation, wastewater management, and pathogen detection. It is equally beneficial for industrial food processing with enhanced food production with excellent market value, elevated nutritional and sensing property, improved safety, and better antimicrobial protection. Nanotechnology can also reduce post-farming losses by increasing the shelf life with the aid of nanoparticles. However, further investigation is required to solve the safety and health risks associated with the technology.

Introduction

     The foremost universal challenge on our planet is the question of establishing food security for a rapidly increasing population in the world. Predictions show that food demand is likely to rise from 59 to 98% for the world population reaching 9 billion by 2050 (Duro et al., 2020). Despite an increase of the world population particularly in developing countries, the global food supply interrupted by the expenditure of bio-resources for production of energy, manufacturing chemicals, high post farming loss, less value addition, inefficient distribution and marketing systems, and other factors (Barrett, 2021; Sekhon, 2014). Farmers throughout the world will focus on using new innovations and technologies for enhancing the production of crops through intensive and extensive agriculture. The current efforts further boosted through the use of nano-modified stimulants and precision farming. Agricultural efficiency, soil improvement, secure water use, distribution of food in stores, and its quality are basic factors of securing food that may be improved via advances in nanotechnology research (Ashraf et al., 2021; Sastry et al., 2011).

     Newer technology that will increase the production and reduce food wastage is important to maintain sustainable living standards of the nation and improve food security. Nanotechnology can provide a path for producing foods with outstanding quality in highly improved workable form along with induction of nutrients bioavailability. Many research investigations are focusing on increasing the application of nanotechnology for the production of crop and food processing (Abobatta, 2018; Axelos and Van De Voorde, 2017; Dasgupta et al., 2015; Peters et al., 2016). Increment in articles, intellectual property, and patents in nano-agriculture-based food with fresh research tendencies in the processing of food, nutraceutical distribution, packing, quality control, and serviceable food is a highly expanding field in nanotechnology research (Dasgupta et al., 2015).

Conclusions

     Nanotechnology is a newly emerging, but highly expanding technology in many fields related to human activities and benefits worldwide. Its fascinating phenomena have been witnessed through several research findings that the nanoparticles and nanostructure improve various properties due to small size, larger surface area, and highly catalytic nature. Nanotechnology is crucial in achieving food security, especially in the agriculture sector. It can improve crop production by effective microbial, pest, and weed control with high economic value, security, and safety. It also plays a key role in food processing, food modification, stability, sensing, prolonged shelf life, minimized food losses, and provide safe food. Nanotechnology also minimizes post-harvest losses with better stability, safety, and packaging materials. Nanoparticles like Ag, Au, Zn, TiO2, ZnO, SiO2, and MgO commonly used in food processing, may also induce health risks due to easy penetration through the cell for adverse reactions within various humans, and animal organs as well as parts of plants. Such risks due to nanoparticles or nanocomposite could be controlled in future research via using greener synthesis and searching for easy and cheaper protocols for degradation and removal of existing nanomaterial from the sites of attack.

Abstract

Keywords

1. Introduction

2. MFNPs-based magnetoresponsive effects for biomedical applications

3. Conclusion and perspectives

Declaration of competing interest

Acknowledgments

References

Abstract

One of the most attractive features of magnetic ferrite nanoparticles (MFNPs) in biomedical application is that they can mediate external magnetic field to produce local magnetic field, magnetic thermal and magnetic force effects. These generated effects can later be utilized in the diagnosis and treatment of various diseases. The application performance is mainly determined by the nano-magnetism of MFNPs. Therefore, by modulating the magnetic properties, the improved magnetic resonance (MR) signals, magnetothermal, and magnetomechanical effects of the MFNPs can be achieved. In this review, we summarize the strategies used in the engineering of MFNPs to enhance MR imaging sensitivity and magnetic thermal conversion efficiencies. We will also discuss the detailed magnetoresponsive mechanism arising from the critical magnetic properties of MFNPs. Furthermore, we will highlight the recent progresses of the engineered MFNPs in biomedical applications, with emphasis in MR signal amplification, magnetothermal, and magnetomechanical response in biomedical applications.

1. Introduction

Since ancient Greek time, lodestone, i.e., bulk magnetite (Fe3O4), has been used in the treatment of common ailments. With the advancement in nano-biotechnology, the applications of such magnetic ferrite nanoparticles (MFNPs) have evolved, which extends to the modern biomedicine and clinical study [[1], [2], [3], [4], [5], [6], [7]]. As biological tissues are transparent to magnetic fields, the most attractive advantage of MFNPs in biomedical applications is that they can complete various functions triggered by invisible, tissue-penetrating magnetic field [8,9]. For instance, natural magnetotactic bacteria build iron oxide nanoparticles (IONPs) in a chain configuration to generate a permanent dipole in their cells so as to navigate toward their favorable habitats by sensing the Earth’s magnetic field [10]. These MFNPs possess good biocompatibility, which have already been used as imaging and therapeutic agents in preclinical and clinical settings [2,3,11]. As a result of the excellent magnetoresponsive properties of MFNPs, numerous exciting in vivo biomedical applications are created. These include imaging for non-invasive diagnosis of diseases [12], treatment of diseases with hyperthermia [13], magnetically driven nanorobots [14], magnetic activation of ion channel for cell signaling [15], manipulation of cell’s function and fate [16,17], and so on. Despite the good establishment of these concepts, there is a significant challenge in obtaining effective MFNPs that can achieve full functionality via the efficient conversion of the external energy of magnetic field. As such, due to their poor conversion ability, this greatly hinders their transition from bench to bed.

1- Introduction

2- pH-responsive polymeric nanocarriers

3- Temperature-responsive polymeric nanocarriers

4- Redox potential-responsive polymeric nanocarriers

5- Enzyme-responsive polymeric nanocarriers

6- Light-responsive polymeric nanocarriers

7- Dual-responsive polymeric nanocarriers

8- Future perspectives

9- Conclusions

References

Abstract

In the last decade, considerable attention has been devoted to the use of biodegradable polymeric materials as potential drug delivery carriers. However, bioavailability and drug release at the disease site remain uncontrollable even with the use of polymeric nanocarriers. To address this issue, successful methodologies have been developed to synthesize polymeric nanocarriers incorporated with regions exhibiting a response to stimuli such as redox potential, temperature, pH, and light. The resultant stimuli-responsive polymeric nanocarriers have shown tremendous promise in drug delivery applications, owing to their ability to enhance the bioavailability of drugs at the disease site. In such systems, drug release is controlled in response to specific stimuli, either exogenous or endogenous. This review reports recent advances in the design of stimuli-responsive nanocarriers for drug delivery in cancer therapy. In particular, the synthetic methodologies investigated to date to introduce different types of stimuli-responsive elements within the biomaterials are described. The sufficient understanding of these stimuli-responsive nanocarriers will allow the development of a better drug delivery system that will allow us to solve the challenges encountered in targeted cancer therapy.

Conclusions

In this review, it was focused on the most recent advances in the development of stimuli-responsive nanocarriers for drug delivery in cancer therapy. These smart drug delivery systems respond to the distinct changes in cancer cells, such as changes in pH gradient and elevated secretion of certain enzymes, rather than the conditions in normal cells. Targeting tumors with stimuliresponsive nanocarriers could not only increase the therapeutic benefit and minimize associated toxicity, but could also enhance the curative effect by specifically releasing the anti-cancer drug in a powerful precise mode, both temporally and spatially. The different approaches adopted to incorporate stimuli responsive elements within the polymeric nanocarriers were discussed, with a particular focus on polymeric materials with stimuliresponsive mechanisms in response to pH, redox potential, enzymes, temperature, and light. Despite the tremendous progress that has been made regarding the engineering of new stimuli-responsive materials, several challenges still remain to be addressed with respect to nanomedicine applications. For example, their biodegradability and biocompatibility profiles should be critically investigated prior to utilization in human clinical trials. Many of these systems have only been reported as an in vitro proof-of-concept and follow-up work in vivo preclinical models has been described for only a few. However, the translation of these stimuli-responsive delivery systems from the bench to the bedside might be facilitated, to some extent, if the regulatory requirements for human clinical trials are considered in light of the key features that render a biopolymer suitable for biomedical application, such as biocompatibility, biodegradability, high drug loading capability, programmable release, excellent in vivo stability, non-cytotoxicity, and ability to support effective targeting. Given the ongoing developments in the field of bionanotechnology along with the wide knowledge accumulated over recent years, it is convinced that the collaborative efforts of chemists, biologists, and medicinal and pharmaceutical scientists will revolutionize the design of responsive polymeric materials for cancer therapy to significantly improve both the quality and duration of the lives of patients with cancer.

1- A brief history of CRISPR

2- CRISPR/Cas9: biology, functionality, and applications

3- Therapeutic targets for ex vivo cell-based CRISPR/Cas9 genome editing

4- CRISPR therapy in clinical trials

5- Challenges and opportunities

References

Abstract

The recently developed CRISPR/Cas9 technology has revolutionized the genome engineering field. Since 2016, increasing number of studies regarding CRISPR therapeutics have entered clinical trials, most of which are focusing on the ex vivo genome editing. In this review, we highlight the ex vivo cell-based CRISPR/Cas9 genome editing for therapeutic applications. In these studies, CRISPR/Cas9 tools were used to edit cells in vitro and the successfully edited cells were considered as therapeutics, which can be introduced into patients to treat diseases. Considering a large number of previous reviews have been focused on the CRISPR/Cas9 delivery methods and materials, this review provides a different perspective, by mainly introducing the targeted conditions and design strategies for ex vivo CRISPR/Cas9 therapeutics. Brief descriptions of the history, functionality, and applications of CRISPR/Cas9 systems will be introduced first, followed by the design strategies and most significant results from previous research that used ex vivo CRISPR/Cas9 genome editing for the treatment of conditions or diseases. The last part of this review includes general information about the status of CRISPR/Cas9 therapeutics in clinical trials. We also discuss some of the challenges as well as the opportunities in this research area.

A brief history of CRISPR

The term CRISPR, or the clustered regularly interspaced palindromic repeats, was first used by Jansen et al., in 2002 to describe a novel family of repetitive DNA sequences presented in the genomes of prokaryotes [1]. These unusual repeated sequences were first detected in 1987 by Nakata et al. in Escherichia coli [2], and then recognized to be widespread in archaea and bacteria by Mojica et al., in 2000 [3]. After Jansen et al. identified the CRISPR-associated (Cas) genes in 2002 [1], Mojica et al. [4], Vergnaud et al. [5], and Bolotin et al. [6] revealed the exrtachromosomal and phage-associated origins of the spacers that separate the individual direct repeats, in 2005. Speculations about CRISPR arrays as immune memory and defense mechanism against virus envisions were then proposed [6,7], while the first experimental evidence for CRISPR/Cas system based adaptive immunity was reported by Horvath et al., in 2007 [8]. Following the series discoveries on the basic function and mechanism of CRISPR systems [9–12], in 2010 Moineau et al. demonstrated the Cas9 enzyme (formerly named Cas5, COG3513, Csn1 or Csx12), which is guided by spacer sequences, cleaves target DNA [13]. In 2012, Doudna, Charpentier et al. [14], and Siksnys et al. [15] proved that Streptococcus pyogenes and Streptococcus thermophiles Cas9 can be guided by CRSIPR RNAs (crRNAs) to cleave target DNA in vitro, through forming a double-strand break (DSB). In 2013, Zhang et al. [16], Church et al. [17], and Doudna et al. [18] independently demonstrated the applications of engineered CRISPR/Cas9 systems for genome editing in mammalian cells. This represented a giant leap forward in the genome engineering field and triggered a tremendous number of studies regarding the use of the CRISPR/Cas9 platform for eukaryotic gene editing in a wide range of species, including flies [19,20], zebrafish [21–24], frogs [25], mice [26–28], rats [26,29], pigs [30], and monkeys [31,32].

Discussion

Methods

References

Discussion

In the present work, we describe a fast, sensitive and easy-toimplement approach for the identification of artificial, ligandresponsive riboswitches in human cells in a high-throughput manner. By applying cDNA-amplicon-seq-based counting of conditionally expressed mRNAs, functional riboswitch candidates can be rapidly identified from complex libraries containing several thousand constructs, thereby enabling the exploration of large sequence spaces. Using this method, we identified tetracyclineand guanine-inducible hammerhead and HDV riboswitches, allosterically controllable Twister-riboswitches for use in human cells and finally, switches using a mode of action based on the conditional modulation of U1-snRNP-dependent polyadenylation. Our initial approach was based on the fact that functional aptazyme riboswitch sequences within a gene expression construct lead to the conditional, ligand-dependent cleavage and subsequent cellular degradation of the mRNA in which they are encoded. Due to the mRNA-intrinsic mode of action, the abundance and identity of riboswitch candidate sequences is directly linked to their function. A similar concept was previously applied to identify enhancer elements on a whole-genome scale, by cloning enhancer candidate libraries into reporter plasmids and quantifying the self-amplified reporter mRNAs by amplicon-seq. This method is known as “self-transcribing active regulatory region sequencing” (STARR-seq)22. Recently, efforts have also been undertaken in the field of ribozyme and riboswitch engineering, to establish high-throughput screening methods. Townshend and colleagues developed a method allowing screening of construct libraries in yeast10. However, this method relied on FACS-selection to generate several bins of low- to high-expressing constructs prior to the sequencing-based identification of functional sequences. Moreover, their method required expensive sequence barcoding and has not yet been shown to be transferrable to human cells. More recently, the Yokobayashi group published a series of papers, utilizing sequencing of in vitrotranscribed constructs to analyze cleavage of Twister-, HDV-like drz-Agam-2-1- and Pistol ribozyme variants23–25. They further described a strategy for the identification of functional constructs in cells11. However, that method requires the isolated RNA to be separated into a non-cleaved and cleaved fraction by gel electrophoresis prior to sequencing, which again, is labor-intensive, more difficult to implement and potentially error-prone. In contrast, the method described in our work enables the rapid identification of conditionally cleaving aptazymes in a relevant cellular system, like e.g. the human cell line HEK-293, in a high-throughput manner, independent of additional pre-selection steps and costly sequence barcoding. The method relies on five core steps, namely library transfection, RNA extraction, cDNA synthesis, PCR amplification and sequencing, and enables direct screening for mRNA selfcleavage in libraries harboring up to approximately 18,000 constructs. Interestingly, while preparing our manuscript, the Smolke lab published a similar method that also successfully applies cDNA-sequencing to identify riboswitches26. In contrast to our method, which exploits the inherent self-barcoding nature of permutated sequence, the method described by Xiang et al. requires an additional sequence barcoding step26. Moreover, while both approaches underscore the applicability and value of cDNAamplicon-sequencing for the discovery of gene switches based on ribozyme platforms, the data presented in our study further extend the use of this method towards expression platforms beyond ribozymes, as demonstrated by the U1-snRNP library screen.

1- Introduction

2- Multi-layer NPs for CRISPR-Cas9 delivery

3- Novel building blocks for multi-layer NPs

4- Conclusions and perspective

References

Abstract

The clustered regularly interspaced short palindromic repeat (CRISPR) has great potential to revolutionize biomedical research and disease therapy. The specific and efficient genome editing strongly depends on high efficiency of delivery of the CRISPR payloads. However, optimization of CRISPR delivery vehicles still remains a major obstacle. Recently, various non-viral vectors have been utilized to deliver CRISPR tools. Many of these vectors have multi-layer structures assembled. In this review, we will introduce the development of CRISPR-Cas9 systems and their general therapeutic applications by summarizing current CRISPR-Cas9 based clinical trials. We will highlight the multi-layer nanoparticles (NPs) that have been developed to deliver CRISPR cargos in vitro and in vivo for various purposes, as well the potential building blocks of multi-layer NPs. We will also discuss the challenges in making the CRISPR tools into viable pharmaceutical products and provide potential solutions on efficiency and biosafety issues.

Introduction

In recent years, a series of programmable DNA nuclease-dependent genome editing techniques have been developed to enable efficient genetic manipulation in eukaryotes, particularly in mammalian cells [1-3]. These genome-editing techniques used for gene knock-out or knock-in predominately include meganucleases, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR-Cas system [4-7]. These technologies all employ endonucleases to induce double strand breaks (DSB) at intended sites in the genome, followed by DSB repair through different mechanisms including non-homologous end joining (NHEJ), homologous recombination (HR), homology-mediated end joining (HMEJ) and microhomology-mediated end joining (MMEJ) [8]. Among these genome editing techniques, CRISPR-Cas9 is a gene editing system derived from the bacterial adaptive immune system, which is capable of combating the invasive viral genome under the guidance of short sequence RNA. CRISPR-Cas9 system is highly versatile because of its simplicity and high efficiency, moreover, any genome sequence theoretically can be edited by this system. In principle, the whole genome can be site-specifically modified at the genetic level in cells and tissues with CRISPR-Cas9 system, which further allows researchers to study the relationship between gene mutations and biological phenotypes. CRISPR-Cas9 system has driven innovative applications from basic biology research to biotechnology and medicine [9].

1- Introduction

2- Materials and methods

3- Results and discussion

4- Conclusion

References

Abstract

In this paper, we report on spatial redistribution of bone marrow mesenchymal stem cells loaded with magnetic nanoparticles under the influence of continuously applied magnetic field. Semiconductor nanoparticles were synthesized by epitaxial growth of a GaN thin layer on magnetic sacrificial core consisting of ZnFe2O4 nanoparticles. Different quantities of nanoparticles were incubated in vitro with mesenchymal stem cells. High density of nanoparticles (50 μg/ml) leads to a decrease in the number of cells during incubation, while the density of nanoparticles as low as 10 μg/ml is enough to drag cells in culture and rearrange them according to the spatial distribution of the magnetic field intensity.

Introduction

Stem cells are defined by their ability to self-renewal and differentiation into more specialized cells, depending on their level of potency [1]. Being easily harvested and cultured, Mesenchymal Stem Cells (MSC) are among the most used stem cells in regenerative medicine. MSC can differentiate into bone, cartilage, fat, tendon, and other different cell lines depending on the given conditions and growth factors [2]. MSC therapeutic potential is enormous and covers a wide medical area, from bone marrow transplantation in blood cancers [3] to the treatment of various diseases like osteoarthritis [4], Crohn`s disease [5], diabetes [6], cardiovascular diseases [7], otorhinolaryngologic pathologies [8], etc. There are several major issues in the therapeutic processes including the non-invasive cellular imaging in high quality transport of therapeutic agent, as well as the remote manipulation in the desired area. The success rate of the therapy based on stem cells depends on the accuracy the cells are delivered to the site of interest. Magnetic resonance imaging (MRI) represents an important tool in cells tracking [9]. The MRI usually includes gadolinium-based contrast agents. Over the last years, however, different types of paramagnetic nanoparticles have been used for tracking cells [10]. The advantage of using nanoparticles for cells tracking relies on the possibility of tuning with the material concentration and chemical composition. Besides tracking, nanoparticles offer the possibility to manipulate with cells and control their spatial position in vivo [11]. The superparamagnetic iron oxide (SPIO), usually stabilized by coating with dextran, gelatine, chitosan or other polymers, is among the most commonly used nanoparticles for cells tracking [12, 13]. Our previous investigations have shown that uncoated gallium nitride nanoparticles (GaN) do not affect the viability and proliferation of endothelial cells [14] and can be used for multifunctional therapeutic purposes which include cells tracking [15]. The use of piezoelectric nanoparticles in combination with magnetic ones could increase the impact of smart materials not only in remote cells imaging but also in the control of the cellular metabolic activity [16-19].

DNA methylation is an epigenetic factor, which plays important roles in embryo and many other diseases development. This factor determines gene expression, and when half of them have CpG islands, DNA methylation and its enzyme effectors have been under the vast studies. Whole genome DNA demethylation is a crucial step of embryogenesis and also cell fate determination in embryos. Therefore, demethylation agents were used as a tool for dedifferentiation and transdifferentiation. Although many of these efforts have been successful, but using this method gave us a vast spectral cell type which is confusing. In this article, we briefly reviewed DNA methylation, and its role in embryogenesis and gene expression. In addition to that, we introduce studies that used this action as a direct method in induction of stem cells and cell fate decision.

Introduction

It has been shown that, epigenetic factors determine transcriptome of cells. These factors determine cell functions and identities [1]. Cell fate determination is among the most significant aspects influenced by these factors [2]. DNA methylation occurring on cytosine in mammals is a major epigenetic factor, which its presence and absence may pose control over 50% of genes, especially developmental-related genes [3]. It has been hypothesized that; DNA demethylation is one of the most significant events in initiation of embryo development after fertilization, which has been named as Zygote Gene Activation (ZGA) [4]. This procedure induces expression of many stemness-related genes. Recently, many studies showed role of DNA demethylation and other epigenetic factors in changing cell fate [2]. It has been proved that, DNA demethylation could change fate of specific somatic cells to another somatic identity in a process named as transdifferentiation [5]. This phenomenon can reprogram somatic cells directly to stem cells [6]. DNA methylation enzyme effectors can establish new fates in differentiation process of stem cells as well [7]. Although it is believed that, disruption of epigenome or reactivation of developmental process in demethylation process could be a potential answer to these phenomena, but related findings have shown that, different roles of DNA demethylation are very confusing to determine. Furthermore, the basic concept and application of this field have been hindered because of its reproducibility and cell fate heterogeneity.