Increasing vaccine production using pulsed ultrasound waves.

Vaccination is a safe and effective approach to prevent deadly diseases. To increase vaccine production, we propose that a mechanical stimulation can enhance protein production. In order to prove this hypothesis, Sf9 insect cells were used to evaluate the increase in the expression of a fusion protein from hepatitis B virus (HBV S1/S2). We discovered that the ultrasound stimulation at a frequency of 1.5 MHz, intensity of 60 mW/cm2, for a duration of 10 minutes per day increased HBV S1/S2 by 27%. We further derived a model for transport through a cell membrane under the effect of ultrasound waves, tested the key assumptions of the model through a molecular dynamics simulation package, NAMD (Nanoscale Molecular Dynamics program) and utilized CHARMM force field in a steered molecular dynamics environment. The results show that ultrasound waves can increase cell permeability, which, in turn, can enhance nutrient / waste exchange thus leading to enhanced vaccine production. This finding is very meaningful in either shortening vaccine production time, or increasing the yield of proteins for use as vaccines.

RANKL release from self-assembling nanofiber hydrogels for inducing osteoclastogenesis in vitro.

PURPOSE: To develop a nanofiber hydrogel (NF-hydrogel) for sustained and controlled release of the recombinant receptor activator of NF-kB ligand; (RANKL) and to characterize the release kinetics and bioactivity of the released RANKL. METHODS: Various concentrations of fluorescently-labelled RANKL protein were added to NF-hydrogels, composed of Acetyl-(Arg-Ala-Asp-Ala)4-CONH2 [(RADA)4] of different concentrations, to investigate the resulting in vitro release rates. The nano-structures of NF-hydrogel, with and without RANKL, were determined using atomic force microscopy (AFM). Released RANKL was further analyzed for changes in secondary and tertiary structure using CD spectroscopy and fluorescent emission spectroscopy, respectively. Bioactivity of released RANKL protein was determined using NFATc1 gene expression and tartrate resistant acid phosphatase (TRAP) activity of osteoclast cells as biomarkers. RESULTS: NF-hydrogel concentration dependent sustained release of RANKL protein was measured at concentrations between 0.5 and 2%(w/v). NF-hydrogel at 2%(w/v) concentration exhibited a sustained and slow-release of RANKL protein up to 48h. Secondary and tertiary structure analyses confirmed no changes to the RANKL protein released from NF-hydrogel in comparison to native RANKL. The results of NFATc1 gene mRNA expression and TRAP activities of osteoclast, showed that the release process did not affect the bioactivity of released RANKL. CONCLUSIONS: This novel study is the first of its kind to attempt in vitro characterization of NF-hydrogel based delivery of RANKL protein to induce osteoclastogenesis. We have shown the self-assembling NF-hydrogel peptide system is amenable to the sustained and controlled release of RANKL locally; that could in turn increase local concentration of RANKL to induce osteoclastogenesis, for application to the controlled mobilization of tooth movement in orthodontic procedures. STATEMENT OF SIGNIFICANCE: Orthodontic tooth movement (OTM) occurs through controlled application of light forces to teeth, facilitating the required changes in the surrounding alveolar bone through the process of bone remodelling. The RANKL system regulates alveolar bone remodelling and controls root resorption during OTM. The use of exogenous RANKL to accelerate OTM has not been attempted to date because large quantities of RANKL for systemic therapy may subsequently cause serious systemic loss of skeletal bone. The controlled and sustained local release of RANKL from a carrier matrix could maximize its therapeutic benefit whilst minimizing systemic side effects. In this study a NF-hydrogel was used for sustained and controlled release of RANKL and the release kinetics and biofunctionality of the released RANKL was characterized. Our results provide fundamental insight for further investigating the role of RANKL NF-hydrogel release systems for inducing osteoclastogenesis in vivo.

Pulsed ultrasound for enhancing vaccine production.

Hepatitis B is an infectious liver disease and vaccination is an effective way to protect individuals. We have applied mechanical wave stimulation to increase protein production. To validate our design, we used Sf9 insect cells to increase antigen fragment fusion protein expression for hepatitis B virus (HBV S1/S2). We discovered that stimulation at a frequency of 1.5 MHz, intensity of 60 mW/cm(2), for a duration of 10 minutes per day increased HBV S1/S2 production by 15%. This finding is very significant for shortening vaccine production time or increasing the yield of proteins for use as vaccines.

Pegylated glucose gold nanoparticles for improved in-vivo bio-distribution and enhanced radiotherapy on cervical cancer.

Pharmacokinetics and bio-distribution are crucial factors affecting the performance of an intravenous drug. In this study, we explore the combined use of glucose and polyethylene glycol (PEG) ligands to further improve gold nanoparticle (GNP) pharmacokinetics and bio-distribution, with the aim of using the drug for in-vivo radiotherapy. The inclusion of PEG was found to significantly prolong the half-life period, where PEG-Glu-GNPs achieved 6.17 +/- 3.71 h, compared to 1.23 +/- 0.14 h for Glu-GNPs and 1.07 +/- 0.22 h for uncoated GNPs. Our data indicates that nanoparticle size impacts cell uptake performance, with 20 nm being the optimal diameter for cancer treatment applications. Although PEG-Glu-GNPs mainly distributed in the spleen, liver, lung, and kidneys, the concentration of PEG-Glu-GNPs in tumour tissue was 20 times higher than healthy cells in the uterus and ovaries, reaching 9.22 +/- 2.41 microg/g cancer tissue at 48 h after injection. This difference in uptake holds promise for selective tumor targeting which can in turn lead to more effective radiotherapy through the interaction of X-rays and GNPs. Specifically tumor size after 47 days of treatment had reduced to (769 +/- 92) mm3 compared to (1432 +/- 269) mm3 using X-rays alone and (3514 +/- 1818) mm3 without any treatment. Moreover, the mice remained healthy without statistically significant weight loss. Results of our pharmacokinetic and bio-distribution study as well as therapeutic data for PEG-Glu-GNPs in our tumor bearing animal model demonstrate that PEG-Glu-GNPs provide excellent in-vivo stability, tumor targeting function, and radiotherapeutic enhancement effects, providing useful insights for further clinical studies.

Applications of low-intensity pulsed ultrasound to increase monoclonal antibody production in CHO cells using shake flasks or wavebags.

Many technologies, such as cell line screening and host cell engineering, culture media optimization and bioprocess optimization, have been proposed to increase monoclonal antibody (mAb) production in Chinese Hamster Ovary (CHO) cells. Unlike the existing biochemical approaches, we investigated stimulation using low-intensity pulsed ultrasound (LIPUS) as a purely physical approach, offering enhanced scalability, contamination control and cost-efficiency, while demonstrating significantly increased cell growth and antibody production. It was found that daily ultrasound treatments at 40 mW/cm(2) for 5 min during cell culture increased the production of human anti-IL-8 antibody by more than 30% using 10 or 30 mL shake flasks. Further increasing the ultrasound dosage (either intensities or the treatment duration) did not appreciably increase cell growth or antibody production, however feeding the culture with additional highly-concentrated nutrients, glucose and amino acids (glutamine in this case), did further increase cell growth and antibody titer to 35%. Similar ultrasound treatments (40 mW/cm(2), 5 min per day) when scaled up to larger volume wavebags, resulted in a 25% increase in antibody production. Increased antibody production can be attributed to both elevated cell count and the ultrasound stimulation. Theoretical study of underlying mechanisms was performed through the simulations of molecular dynamics using the AMBER software package, with results showing that LIPUS increases cell permeability. The significance of this study is that LIPUS, as a physical-based stimulation approach, can be externally applied to the cell culture without worrying about contamination. By combining with the existing technologies in antibody production, LIPUS can achieve additional mAb yields. Because it can be easily integrated with existing cell culture apparatuses, the technology is expected to be more acceptable by the end users.

Developing trends in aptamer-based biosensor devices and their applications.

Aptamers are, in general, easier to produce, easier to store and are able to bind to a wider variety of targets than antibodies. For these reasons, aptamers are gaining increasing popularity in environmental monitoring as well as disease detection and disease management applications. This review article examines the research and design of RNA and DNA aptamer based biosensor systems and applications as well as their potential for integration in effective biosensor devices. As single stranded DNA or RNA molecules that can bind to specific targets, aptamers are well suited for biomolecular recognition and sensing applications. Beyond being able to be designed for a near endless number of specific targets, aptamers can also be made which change their conformation in a predictable and consistent way upon binding. This can lead to many unique and effective detection methods using a variety of optical and electrochemical means.

Biomolecule delivery into canola protoplasts by centrifuging cells with microbubbles.

We have successfully delivered FITC and FITC-Dextran (70, 250 kDa) into canola protoplasts by centrifuging cells with different amounts of microbubbles at variable centrifuge speed. The efficiency is around 90%, while cell viability remains high. Confocal microscopy images show that both FITC and FITC-Dextran are scattered inside the cytoplasm and the cell nucleus. Pores are observed on canola protoplast cell membranes and cell walls when centrifuged with microbubbles, while the membrane of cells centrifuged alone remain intact and smooth. We hypothesize that the collision between the microbubbles and cells or the bursting of microbubbles are the main reasons for the formation of these pores. Biomaterials can diffuse into the cells once the pathway is created.

Nanoformulation of paclitaxel to enhance cancer therapy.

UNLABELLED: Paclitaxel is a microtubule inhibitor causing mitotic arrest and is widely used in cancer chemotherapy. However, its poor water solubility restricts its direct clinical applications. In this article, we report paclitaxel-loaded nanoparticles that are water soluble and that can improve the drug's bio-distribution and therapeutic efficacy. Paclitaxel-loaded nanoparticles were synthesized by using Pluronic copolymers (F-68 and P-123) and surfactant (Span 40) as nanocarrier. The toxicity and cellular uptake of paclitaxel-loaded nanoparticles were evaluated. The paclitaxel-loaded nanoparticles can completely disperse into phosphate buffer saline to produce a clear aqueous suspension. Based on HPLC analysis, the drug-loading rate is 9.0 ± 0.1% while drug encapsulation efficiency is 99.0 ± 1.0%. The cytotoxicity assay was performed using breast cancer MCF-7 and cervical cancer Hela cells. For MCF-7 cells, the half maximal inhibitory concentrations (IC50) of paclitaxel-loaded nanoparticles and paclitaxel are 8.5 ± 0.3 and 14.0 ± 0.7 ng/mL at 48 hours and 3.5 ± 0.4 and 5.2 ± 0.5 ng/mL at 72 hours across several runs. IC50 of paclitaxel-loaded nanoparticles and paclitaxel for Hela cells are 5.0 ± 0.3 and 8.0 ± 0.3 ng/mL at 48 hours and 2.0 ± 0.1 and 6.5 ± 0.3 ng/mL at 72 hours. In-vitro studies show that the drug's nanoformulation gives obvious enhancements in the drug's efficiency at killing cancer cells over paclitaxel alone. Materials of the nanocarrier used for nanoformulation are approved with low toxicity according to the result of cell studies. CONCLUSION: paclitaxel-loaded nanoparticles greatly improved the physicochemical properties of paclitaxel without modifying its chemical structure, allowing for deep-site cancer drug delivery and enhancing the drug therapeutic efficiency.

Smart gold nanoparticles enhance killing effect on cancer cells.

The present study explored the cellular uptake dynamics, the subcellular location and the internalization mechanisms of gold nanoparticles (GNPs) and glucose-capped GNPs (Glu-GNPs). The cancer radiotherapy-enhancing effects of GNPs were also evaluated. We synthesized the GNPs and Glu-GNPs by the seeding technique. The effects on cellular uptake and the radiosensitizing effect induced by GNPs and Glu-GNPs at lower doses were investigated using two human cancer cell lines (HeLa and MCF-7). The intracellular location of the nanoparticles was analyzed by transmission electron microscopy (TEM). Analysis of cellular apoptosis following GNP-based radiotherapy was performed by flow cytometry and TUNEL assay. Cancer cells took up more Glu-GNPs than naked GNPs and the uptake curve showed size- and cell-dependent uptake. GNPs were mainly located in the cytoplasm and endocytosis is the mechanism behind the internalization of GNPs and Glu-GNPs. Lower doses of GNPs and Glu-GNPs still enhanced the killing effect using X-ray irradiation, although the apoptotic rate was not altered. The results presented in this study provide evidence that Glu-GNPs may have a bright future in tumor-targeted diagnosis and treatment.

Sputum microRNA profiling: a novel approach for the early detection of non-small cell lung cancer.

PURPOSE: MicroRNAs (miRNAs) post-transcriptionally regulate hundreds of gene targets involved in tumorigenesis thereby controlling vital biological processes, including cellular proliferation, differentiation and apoptosis. MiRNA profiling is an emerging tool for the potential early detection of a variety of malignancies. This study was conducyed to assess the feasibility and methodological robustness of quantifying sputum miRNAs, employing quantitative real-time polymerase chain reaction (RT-qPCR) and cluster analysis on an optimized miRNA profile as a novel approach for the early detection of non-small cell lung cancer (NSCLC). METHODS: The relative expressions of 11 miRNAs in sputum (miR-21, miR-145, miR-155, miR-205, miR-210, miR-92, miR-17-5p, miR-143, miR-182, miR-372, and let-7a) in addition to U6 were retrospectively assessed in four NSCLC-positive and four negative controls. Subsequently, a set of five miRNAs (miR-21, miR-143, miR-155, miR-210, miR-372) was selected because of degree of relatedness observed in the cluster analysis and tested in the same sputum sample set. The five optimized miRNAs accurately clustered these eight retrospective patients into NSCLC positive cases and negative controls. The five miRNA panel was then prospectively quantified in the sputum of 30 study patients (24 NSCLC cases and six negative controls) in a double-blind fashion to validate a five miRNA panel using hierarchical cluster analysis. RESULTS: The optimized five miRNA panel detected NSCLC (83.3% sensitivity and 100% specificity) in 30 prospectively accrued study patients. CONCLUSION: Sputum miRNA profiling using cluster analysis is a promising approach for the early detection of non-small cell lung cancer. Further investigation using this approach is warranted.

Ultrasound-enhanced monoclonal antibody production.

With the rapidly growing demand for monoclonal antibody (mAb)-based products, new technologies are urgently needed to increase mAb production while reducing manufacturing costs. To solve this problem, we report our research findings of using low-intensity pulsed ultrasound (LIPUS) to enhance mAb production. LIPUS with frequency of 1.5 MHz and pulse repetition frequency of 1 kHz, as well as duty cycle of 20%, was used to stimulate hybridoma cells to enhance the production of mAb, anti-CD4 (hybridoma GK1.5). The enzyme-linked immunosorbent assay results show a 60.42 ± 7.63% increase of mAb expression in hybridoma cells. The evidence of structural changes of the cellular outer membrane in both transmission electron microscopy and scanning electron microscopy images and the more than 20% lactate dehydrogenase release indicates that the increased mAb production is related to the increased cell permeability induced by LIPUS. This value-added ultrasound technology provides a potential cost-effective solution for pharmaceutical companies to manufacture mAb-based drugs. The technology, in turn, can reduce the drug manufacturing costs and decrease health care spending.

FITC delivery into plant cells using magnetic single-walled carbon nanotubes.

In this paper, fluorescein isothiocyanate (FITC) was covalently bonded with magnetic single-walled carbon nanotubes (mSWCNTs) that were purified using our previous method. To demonstrate our design, mSWCNT-FITC was delivered into plant cells (canola and carrot cells) driven by external magnetic forces. From FACS results, the FITC delivery efficiency was about 100% for both two canola and carrot protoplasts, which were further confirmed by the confocal and sectional TEM images. Some mSWCNTs were found trapped both inside the endosomes of canola protoplast and outside endosome near the nuclear membrane of carrot protoplast according to the sectional TEM images. All results showed that mSWCNT is a good delivery carrier for biomolecules.

High-throughput quantitative analysis with cell growth kinetic curves for low copy number mutant cells.

The mutation rate in cells induced by environmental genotoxic hazards is very low and difficult to detect using traditional cell counting assays. The established genetic toxicity tests currently recognized by regulatory authorities, such as conventional Ames and hypoxanthine guanine phosphoribosyl-transferase (HPRT) assays, are not well suited for higher-throughput screening as they require large amounts of test compounds and are very time consuming. In this study, we developed a novel cell-based assay for quantitative analysis of low numbers of cell copies with HPRT mutation induced by an environmental mutagen. The HPRT gene mutant cells induced by the mutagen were selected by 6-thioguanine (6-TG) and the cell's kinetic growth curve monitored by a real-time cell electronic sensor (RT-CES) system. When a threshold is set at a certain cell index (CI) level, samples with different initial mutant cell copies take different amounts of time in order for their growth (or CI accumulation) to cross this threshold. The more cells that are initially seeded in the test well, the faster the cell accumulation and therefore the shorter the time required to cross this threshold. Therefore, the culture time period required to cross the threshold of each sample corresponds to the original number of cells in the sample. A mutant cell growth time threshold (MT) value of each sample can be calculated to predict the number of original mutant cells. For mutagenesis determination, the RT-CES assay displayed an equal sensitivity (p > 0.05) and coefficients of variation values with good correlation to conventional HPRT mutagenic assays. Most importantly, the RT-CES mutation assay has a higher throughput than conventional cellular assays.

Pharmacokinetic and toxicological evaluation of multi-functional thiol-6-fluoro-6-deoxy-D-glucose gold nanoparticles in vivo.

We synthesized a novel, multi-functional, radiosensitizing agent by covalently linking 6-fluoro-6-deoxy-D-glucose (6-FDG) to gold nanoparticles (6-FDG-GNPs) via a thiol functional group. We then assessed the bio-distribution and pharmacokinetic properties of 6-FDG-GNPs in vivo using a murine model. At 2 h, following intravenous injection of 6-FDG-GNPs into the murine model, approximately 30% of the 6-FDG-GNPs were distributed to three major organs: the liver, the spleen and the kidney. PEGylation of the 6-FDG-GNPs was found to significantly improve the bio-distribution of 6-FDG-GNPs by avoiding unintentional uptake into these organs, while simultaneously doubling the cellular uptake of GNPs in implanted breast MCF-7 adenocarcinoma. When combined with radiation, PEG-6-FDG-GNPs were found to increase the apoptosis of the MCF-7 breast adenocarinoma cells by radiation both in vitro and in vivo. Pharmacokinetic data indicate that GNPs reach their maximal concentrations at a time window of two to four hours post-injection, during which optimal radiation efficiency can be achieved. PEG-6-FDG-GNPs are thus novel nanoparticles that preferentially accumulate in targeted cancer cells where they act as potent radiosensitizing agents. Future research will aim to substitute the (18)F atom into the 6-FDG molecule so that the PEG-6-FDG-GNPs can also function as radiotracers for use in positron emission tomography scanning to aid cancer diagnosis and image guided radiation therapy planning.

Low-intensity pulsed ultrasound-mediated stimulation of hematopoietic stem/progenitor cell viability, proliferation and differentiation in vitro.

Low-intensity pulsed ultrasound (LIPUS) stimulated the viability, proliferation and differentiation of hematopoietic stem/progenitor cells (HSPC) from fresh and cryopreserved peripheral blood leukapheresis product, as well as cord blood when applied for 10 min each day for 4 days. Cell viability, proliferation and differentiation were assessed on day 5 by viable cell counting, MTS proliferation assay, flow cytometry, and colony-forming unit assay. LIPUS stimulation: (i) enhanced the proliferation of fresh HSPC and maintained the viability of cryopreserved HSPC in vitro; (ii) did not affect the percentage of CD34(+) and CD14(+) cells; and (iii) enhanced burst-forming unit-erythroid colony formation. Hence, we suggest that this novel LIPUS stimulation approach might enhance the efficacy of clinical transplantation and cellular therapies using HSPC.

Applications of ultrasound to enhance mycophenolic acid production.

Reducing production costs for fermentation-based drugs (e.g., antibiotics) is crucial for the long-term sustainability of healthcare. In this study, we propose a novel low-intensity pulsed ultrasound (LIPUS) stimulation scheme using a nominal frequency of 1.5 MHz with a 20% duty cycle (200 µs ultrasound on and 800 µs ultrasound off) to increase production of fermentation-based drugs. We chose Penicillium brevicompactum as a model system to demonstrate the performance of our LIPUS system. Penicillium brevicompactum can produce mycophenolic acid (MPA), an immunosuppressive agent commonly used to prevent rejection after organ transplantation. We have stimulated Penicillium brevicompactum in 50 mL shake flasks using LIPUS during its fermentation. After a series of screening experiments to optimize ultrasound parameters (e.g., ultrasound intensities, treatment duration and treatment frequency per day), it was concluded that ultrasound stimulation can increase MPA production by as much as 60% when treated eight times a day for 10-min durations at an intensity (spatial peak temporal average) of 200 mW/cm(2). These findings elucidate a new approach to reduce the cost of producing fermentation-based drugs.

SN-38 loaded polymeric micelles to enhance cancer therapy.

7-Ethyl-10-hydroxycamptothecin (SN-38) loaded poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (Pluronic F-108) and poly(ethylene glycol)-block-poly(ε-caprolactone) (PEG-b-PCL) nanoparticles were successfully prepared by a modified film hydration method and characterized by scanning electric microscopy (SEM), transmission electron microscopy (TEM), atomic force microscopy (AFM) and dynamic light scattering (DLS). Satisfactory drug loading of 20.73 ± 0.66% and a high encapsulation efficiency of 83.83 ± 1.32% were achieved. The SN-38 nanoparticles (SN-38 NPs) can completely disperse into a phosphate buffered saline (PBS) medium to produce a clear aqueous suspension that remains stable for up to three days. Total drug releases were 67.91% and 91.09% after 24 h in a PBS or fetal bovine serum (FBS) medium. Half maximal inhibitory concentration (IC(50)) tests of SN-38 and SN-38 NPs on A549 lung cells produced results of 200.0 ± 14.9 ng ml(-1) and 80.0 ± 4.6 ng ml(-1), respectively. Similarly, IC(50) tests of SN-38 and SN-38 NPs on MCF-7 breast cells yielded results of 16.0 ± 0.7 ng ml(-1) and 8.0 ± 0.5 ng ml(-1), respectively. These in vitro IC(50) studies show significant (p < 0.01) enhancement of the SN-38 NP drug efficiency in killing cancer cells in comparison to the free drug SN-38 control. All the materials used for this nanoformulation are approved by the US FDA, with the virtue of extremely low toxicity to normal cells.

Efficient and rapid uptake of magnetic carbon nanotubes into human monocytic cells: implications for cell-based cancer gene therapy.

Monocyte-based gene therapies in cancer have been hampered by either the resistance of these cells to non-viral molecular delivery methods or their poor trafficking to the tumor site after their ex vivo manipulations. Magnetic nanoparticles (MNP)-loaded genetically engineered monocytes can efficiently delivered to tumor site by external magnetic field, but they are not ideal delivery tools due to their spherical shape. Hence, we have investigated the cellular uptake efficiency and cytotoxicity of fluorescein isothiocyanate (FITC)-labelled magnetic carbon nanotubes (FITC-mCNT) in human monocytic leukemia cell line THP-1 for application in cell-based gene therapy against cancer. Uptake of FITC-mCNT into THP-1 cells reached 100% only 1 h after the delivery. Confocal imaging confirmed that FITC-mCNT entered the cell cytoplasm and even into the nucleus. FITC-mCNT uptake did not compromise cell viability. This delivery system might therefore enhance cell-based cancer gene therapies.

Magnetic gold nanoparticles: synthesis, characterization and its application in the delivery of FITC into KG-1 cells.

In this article, we report a new method-a sonication method to disperse iron oxide nanoparticles into smaller nanoparticles and make gold ions absorb onto the surface or trapped in the micropores of the iron oxide nanoparticles using sonication action. By using quick reduction of ascorbic acid and post-HCI solution treatment, gold covered magnetic nanoparticles (mGNPs) with spherical morphology and uniform size were synthesized in a water solution. The size of the mGNPs was found to be 20-30 nm. Some ideal mGNPs possessed a core-shell structure. The mGNPs were non-cytotoxic and mGNP-fluorescein isothiocyanate (FITC) can enter KG-1 cells when driven by an external magnetic force, which was confirmed by confocal imaging. The confocal image also showed the FITC inside the KG-1 cells was near the nucleus. The fluorescein isothiocyanate (FITC) delivery efficiency is about 100% according to the flow cytometry results.

A low-cost intracellular delivery system based on microbubble and high gravity field.

In this paper, we developed a low-cost intracellular delivery system based on microbubble and high gravity field. We successfully delivered FITC-Dextran (40kD) into hard-to-deliver THP-1 cells. The results showed that our method achieved high delivery efficiency up to 80%. It was found that the delivery efficiency and cell viability were closely related to the centrifuge speed. We speculated that the burst of microbubbles causes transient pore opening thus increasing the chance of biomolecules entering cells. This fast, low-cost and easy-to-operate protocol is very promising for delivering therapeutic genes and drugs into any cells which do not actively take up extracellular materials. This method is most effective for in-vitro delivery, but after delivery, treated cells might be injected back to human for in-vivo imaging.