Liver metastasis is a major obstacle in treating aggressive cancers, and current therapeutic options often prove insufficient. To overcome these challenges, there has been growing interest in ultrasound-mediated drug delivery using lipid-shelled microbubbles (MBs) and nanobubbles (NBs) as promising strategies for enhancing drug delivery to tumors. Our previous work demonstrated the potential of Doxorubicin-loaded C 3 F 8 NBs (hDox-NB, 280 ± 123 nm) in improving cancer treatment in vitro using low-frequency ultrasound. In this study, we investigated the pharmacokinetics and biodistribution of sonicated hDox-NBs in orthotopic rat liver tumors. We compared their delivery and therapeutic efficiency with size-isolated MBs (hDox-MB, 1104 ± 373 nm). Results showed a similar accumulation of hDox in tumors treated with hDox-MBs and unfocused therapeutic ultrasound (hDox-MB+TUS) and hDox-NB+TUS. However, significantly increased apoptotic cell death in the tumor and fewer off-target apoptotic cells in the normal liver were found upon the treatment with hDox-NB+TUS. The tumor-to-liver apoptotic ratio was elevated 9.4-fold following treatment with hDox-NB+TUS compared to hDox-MB+TUS, suggesting that the therapeutic efficacy and specificity are significantly increased when using hDox-NB+TUS. These findings highlight the potential of this approach as a viable treatment modality for liver tumors. By elucidating the behavior of drug-loaded bubbles in vivo , we aim to contribute to developing more effective liver cancer treatments that could ultimately improve patient outcomes and decrease off-target side effects.

Correct measurement of the shell properties of coated microbubbles (MBs) is essential to understanding and optimizing their response to ultrasound (US) exposure parameters in diagnostic and therapeutic ultrasound. MBs are surrounded by blood; however, the influence of the surrounding medium charges on the MB properties is poorly understood. This study aims to measure the medium charge interactions with MB shells by measuring the frequency-dependent attenuation of the same size MBs in mediums of varying charge density. In-house lipid-coated MBs with C3F8 gas core were made and were isolated to a mean size of 2.35um. MBs were diluted to ~8*10^5 MBs/mL in distilled water (DW), and two different concentrations of phosphate-buffered saline solution (PBS-1x and PBS-10x). The frequency-dependent attenuation of the MBs solutions was measured using an aligned pair of PVDF transducers with a center frequency of 10MHz and 100% bandwidth. The MB shell properties were estimated by fitting the linear equation to experiments. Using a pendant drop tensiometer, the surface tension of mm-size drops was measured inside DW, PBS-1x and PBS-10x. The frequency of the peak attenuation changes at different salinity levels was 13, 7.5 and 6.25MHz in DW, PBS-1x and PBS-10x, respectively. The attenuation peak increased by ~140% with increasing ion density. MBs' estimated shell elasticity decreased by 64% between DW and PBS-1x and 36% between PBS-1x and PBS-10x. Reduction in the shell stiffness is in qualitative agreement with the drop surface tension measurements. The shell viscosity was reduced by ~40% between DW and PBS-1x and 42% between PBS-1x and PBS-10x. The reduction in the stiffness and viscosity is possibly due to the formation of a densely charged layer around the shell, further reducing the effective surface tension on the MBs.

Bioactive molecules and their effects have been influenced by their solubility and administration route. In many therapeutic reagents, the performance of therapeutics is dependent on physiological barriers in the human body and delivery efficacy. Therefore, an effective and stable therapeutic delivery promotes pharmaceutical advancement and suitable biological usage of drugs. In the biological and pharmacological industries, lipid nanoparticles (LNPs) have emerged as a potential carrier to deliver therapeutics. Since studies reported doxorubicin-loaded liposomes (Doxil®), LNPs have been applied to numerous clinical trials. Lipid-based nanoparticles, including liposomes, solid lipid nanoparticles (SLNs), and nanostructured lipid nanoparticles, have also been developed to deliver active ingredients in vaccines. In this review, we present the type of LNPs used to develop vaccines with attractive advantages. We then discuss messenger RNA (mRNA) delivery for the clinical application of mRNA therapeutic-loaded LNPs and recent research trend of LNP-based vaccine development.

Lipid-coated microbubbles (MBs) are used in contrast-enhanced ultrasound (CEUS) imaging and MB enhanced therapeutic ultrasound (US). Understanding the MB behavior and the influence of the surrounding medium on its response to the US is necessary to select the suitable US exposure parameters. The MB lipid coating is often charged, however the influence of the ions in the medium on MB behavior is not fully understood. In this work, the influence of the medium salinity on the pressure-dependent MB behavior is investigated for the first time. MBs of different lipid shell compositions are size isolated to achieve the same size distribution. The MBs linear and pressure-dependent attenuation are measured in deionized water, PBS 1×, PBS 2×, and PBS 10× using a system of aligned PVDF 100% bandwidth transducers with a center frequency of 10 MHz and exposures with peak to peak pressure range of 3–140 kPa. With increasing salinity, the linear resonance frequency decreases up to 50% for conventional lipid shell compositions, and the pressure dependence of the resonance frequency is inhibited. By modifying the shell PEG ratio, the salinity effects can significantly be altered. Moreover, the nonlinear pressure-dependent resonance frequency is restored with applications to increased CEUS.

Purpose: A real-time and non-invasive thermometry technique is essential in thermal therapies to monitor and control the treatment. Ultrasound is an attractive thermometry modality due to its relatively high sensitivity to change in temperature and fast data acquisition and processing capabilities. A temperature-sensitive acoustic parameter is required for ultrasound thermometry in order to track the changes in that parameter during the treatment. Currently, the main ultrasound thermometry methods are based on variation in the attenuation coefficient, the change in backscattered energy of the signal (CBE), the backscattered radio-frequency (RF) echo-shift due to change in the speed of sound and thermal expansion of the medium, and change in the amplitudes of the acoustic harmonics. In this work, an ultrasound thermometry method based on second harmonic CBE (CBEh2) and combined fundamental and second harmonic CBE (CBEcomb) is used to produce 2D temperature maps, detect localized heated region generated by low intensity focused ultrasound (LIFU), and control temperature in the heated region. Materials and methods: Ex vivo pork muscle tissue samples were exposed to localized LIFU heating source and 2D temperature maps were produced from the RF data acquired by a 4.2 MHz linear array probe using a Verasonics Vantage (TM) ultrasound scanner (Verasonics Inc., Redmond, WA) after the exposure. Calibrated needle thermocouples were also placed in the ex vivo tissue sample close to the LIFU focal zone for temperature calibration purposes. The estimated temperature maps were the established echo-shift technique. A tissue motion compensation algorithm was also used to reduce the susceptibility to motion artifacts. Results: 2D temperature maps were generated using CBE of acoustic harmonic and echo-shift techniques. The results show a direct correlation between the CBE of acoustic harmonics and focal tissue temperature for a range of temperatures from 37 degrees C (baseline) to 47 degrees C. Conclusions: The findings of this study show that the CBE of acoustic harmonics technique can be used to non-invasively estimate temperature change in tissue in the hyperthermia temperature range.

This dissertation presents the effect of peptide-modified 10 nm gold nanoparticles (GNPs) with chemotherapeutic drugs, bleomycin and cisplatin, and 2 Gy of 6 MV X-ray irradiation in MDA-MB-231 cells. The GNPs were modified with a peptide sequence containing an ‘RGD’ amino acid motif. Bleomycin binds to the surface of the GNPs through a thiol bond and cisplatin has no known significant interaction with the GNP surface. No significant toxicity was induced by introducing GNPs to MDA-MB-231 cells at the 0.3 nM concentration used throughout this dissertation. The surface modification with ‘RGD’ peptides increased accumulation of the GNP constructs 6~7 fold compared to the unmodified counterparts. There was no significant difference in the accumulation of GNPs in the presence of bleomycin or cisplatin. These results suggest that the presence of chemotherapeutics do not affect the accumulation of peptide modified GNPs into cells. The effect of having GNPs with chemotherapeutics was examined. The presence of GNPs with bleomycin decreased the survival of MDA-MB-231 cells by 18 ± 3 % compared to treatment with the same concentration of free bleomycin. Treating cells with GNPs and cisplatin did not have a significant difference in survival compared to the same concentration of free cisplatin treatment. This suggests that conjugating chemotherapeutics onto the GNPs can result in a more efficient delivery of the drug. If the drug does not bind to the GNP surface, having GNPs in the media does not interfere with the uptake of the drug. The effect of radiosensitization in the presence of GNPs was studied by incubating cells with 0.3 nM GNPs prior to irradiation with 2 Gy of 6 MV X-rays. The survival fraction decreased by 19 ± 6 % compared to the irradiated control condition. Lastly, the triple combined effect of GNPs, chemotherapeutics, and irradiation was investigated. The presence of GNPs had an advantage to the combined chemotherapy and radiation therapy. Based on results from these studies, GNPs can be used in addition to combined chemotherapy and radiation therapy for improved outcomes in cancer treatment

Objective: This work aims to determine whether photoacoustic (PA) thermometry from a commercially available PA imaging system can be used to control the temperature in nanoparticle-mediated thermal therapies. Methods: The PA imaging system was interfaced to obtain PA images while scanning ex-vivo tissue. These images were then used to obtain temperature maps in real-time during heating. Validation and calibration of the PA thermometry were done using a fluoroptic thermometer. This thermometer was also used to develop and tune a software-based proportional integral derivative (PID) controller. Finally, a PA-based PID closed-loop controller was used to control gold nanorod (GNR) mediated laser therapy. Results: The use of GNRs substantially enhanced laser heating; the temperature rise increased 7-fold by injecting a GNR solution with a concentration of 0.029 mg/mL. The control experiments showed that the desired temperature could be achieved and maintained at a targeted location in the ex-vivo tissue. The steady-state mean absolute deviations (MAD) from the targeted temperature during control were between 0.16 $^\circ {\kern-0.70007pt}\text{C}$ and 0.5 $^\circ {\kern-0.70007pt}\text{C}$ , depending on the experiment. Conclusion: It was possible to control hyperthermia treatments using a software-based PID controller and a commercial PA imaging system. Significance: The monitoring and control of the temperature in thermal-based therapies are important for assuring a prescribed temperature to the target tissue while minimizing the temperature of the surrounding healthy tissue. This easily implemented non-invasive control system will facilitate the realization of a broad range of hyperthermia treatments.

Gold nanoparticles (GNPs) have been extensively used in cancer research due to their abilities as anti-cancer drug carriers for chemotherapy and as dose enhancers in radiotherapy. Although most GNP research in the past involved cytoplasm localized GNPs, it is predicted that therapy response can be enhanced if GNPs can be effectively targeted into the nucleus. A strategy for designing a GNP-peptide complex for targeting the nucleus will be presented. Three different sequences of peptides (CKKKKKKGGAGDMFG, CGGRKKRRGRRRAP, CALNN) were conjugated onto GNPs. The first peptide was used to stabilize the complex, the second peptide to enhance uptake into the cell, while the third peptide was used to induce nuclear delivery. With nuclear targeting, more damage can be caused to the DNP of cancer cells upon irradiation. This research will establish a more successful NP-based platform that combines treatment modalities and more effectively approach cancer treatment.

Combined use of chemotherapy and radiation therapy is commonly used in cancer treatment, but the toxic effects on normal tissue are a major limitation. This study assesses the potential to improve radiation therapy when combining gold nanoparticle (GNP) mediated radiation sensitization with chemoradiation compared to chemoradiation alone. Incorporation of GNPs with 2 Gy, 6 MV (megavoltage) radiation resulted in a 19 ± 6% decrease in survival of MDA-MB-231 cells. Monte-Carlo simulations were performed to assess dosimetric differences in the presence of GNPs in radiation. The results show that physics dosimetry represents a small fraction of the observed effect. The survival fraction of the cells exposed to GNPs, cisplatin, and radiation was 0.16 ± 0.007, while cells treated with cisplatin and radiation only was 0.23 ± 0.011. The presence of GNPs resulted in a 30 ± 6% decrease in the survival, having an additive effect. The concentration of the GNPs and free drug used for this study was 0.3 and 435 nM, respectively. These concentrations are relatively lower and achievable in an in vivo setting. Hence, the results of our study would accelerate the incorporation of GNP-mediated chemoradiation into current cancer therapeutic protocols in the near future.

This chapter addresses physicochemical properties that affect Nanoparticle (NP) intracellular behavior using Gold NPs (GNPs) as a model system. The main objective is to outline what is known about the effect of GNP size, shape, and surface properties on cellular uptake and intracellular pathway. The authors propose that the entry of GNPs into cells is related to its effectiveness in applications that favor intracellular localization of such GNPs. The authors also discuss how such properties are used to optimize GNP designs for medical applications. Finally, the authors discuss how GNPs may improve disease diagnosis and treatment. Furthermore, how they may be incorporated or used as alternatives to current treatment options is defined.

Optimizing the interface between nanoparticles (NPs) and the biological environment at various levels should be considered for improving delivery of NPs to the target tumor area. For NPs to be successfully delivered to cancer cells, NPs needs to be functionalized for circulation through the blood vessels. In this study, accumulation of Polyethylene Glycol (PEG) functionalized gold nanoparticles (GNPs) was first tested using in vitro monolayer cells and multilayer cell models prior to in vivo models. A diameter of 10 nm sized GNP was selected for this study for sufficient penetration through tumor tissue. The surfaces of the GNPs were modified with PEG molecules, to improve circulation time by reducing non-specific uptake by the reticuloendothelial system (RES) in animal models, and with a peptide containing integrin binding domain, RGD (arginyl-glycyl-aspartic acid), to improve internalization at the cellular level. A 10–12% accumulation of the injected GNP dose within the tumor was observed in vivo and the GNPs remained within the tumor tissue up to 72 h. This study suggests an in vitro platform for optimizing the accumulation of NP complexes in cells and tissue structures before testing them in animal models. Higher accumulation within the tumor in vivo upon surface modification is a promising outcome for future applications where GNPs can be used for drug delivery and radiation therapy.

Recent developments in nanotechnology has provided new tools for cancer therapy and diagnosis. Among other nanomaterial systems, gold nanoparticles are being used as radiation dose enhancers and anticancer drug carriers in cancer therapy. Fate of gold nanoparticles within biological tissues can be probed using techniques such as TEM (transmission electron microscopy) and SEM (Scanning Electron Microscopy) due to their high electron density. We have shown for the first time that cancer drug loaded gold nanoparticles can reach the nucleus (or the brain) of cancer cells enhancing the therapeutic effect dramatically. Nucleus of the cancer cells are the most desirable target in cancer therapy. In chemotherapy, smart delivery of highly toxic anticancer drugs through packaging using nanoparticles will reduce the side effects and improve the quality and care of cancer patients. In radiation therapy, use of gold nanoparticles as radiation dose enhancer is very promising due to enhanced localized dose within the cancer tissue. Recent advancement in nanomaterial characterization techniques will facilitate mapping of nanomaterial distribution within biological specimens to correlate the radiobiological effects due to treatment. Hence, gold nanoparticle mediated combined chemoradiation would provide promising tools to achieve personalized and tailored cancer treatments in the near future.

Efforts to improve conventional cancer therapies have led to the development of cancer nanomedicine. Nanomedicine is an interdisciplinary field that utilizes nanoparticles (NPs) for medical applications. In the last two decades, NPs of different materials, shapes and sizes have been used for various applications ranging from imaging to therapy and targeted drug delivery. Quantification of NP uptake in cancer cells plays a significant role in the analysis of NP-based applications. Currently, Inductive Coupled Plasma (ICP) based methods are being used for quantification purposes. However, quantifying trace-level concentration of NPs, especially when very small sample volume is available, can be challenging.

Nanoparticles (NPs) can be used to overcome the side effects of poor distribution of anticancer drugs. Among other NPs, colloidal gold nanoparticles (GNPs) offer the possibility of transporting major quantities of drugs due to their large surface-to-volume ratio. This is while confining these anticancer drugs as closely as possible to their biological targets through passive and active targeting, thus ensuring limited harmful systemic distribution. In this study, we chose to use bleomycin (BLM) as the anticancer drug due to its limited therapeutic efficiency (harmful side effects). BLM was conjugated onto GNPs through a thiol bond. The effectiveness of the chemotherapeutic drug, BLM, is observed by visualizing DNA double strand breaks and by calculating the survival fraction. The action of the drug (where the drug takes effect) is known to be in the nucleus, and our experiments have shown that some of the GNPs carrying BLM were present in the nucleus. The use of GNPs to deliver BLM increased the delivery and therapeutic efficacy of the drug. Having a better control over delivery of anticancer drugs using GNPs will establish a more successful NP-based platform for a combined therapeutic approach. This is due to the fact that GNPs can also be used as radiation dose enhancers in cancer research.

Nanoparticles have been used as a platform to improve therapeutic results in medical research. Gold nanoparticles (GNPs) have been extensively studied among other nanoparticles in cancer research due to its property being radio-sensitizing by producing more secondary electrons in response to irradiation. It is predicted that closer the GNPs can get to the DNA, the extra secondary electrons will cause more breaks in the DNA. However, the regular uptake pathway of GNPs into the cell is known to be through receptor mediated endocytosis, where GNPs follow the endo-lyso pathway. An effective design of GNP-peptide complex for nuclear targeting will be presented, where three different peptide sequences are conjugated onto 10 nm sized spherical GNPs. An effective design of a GNP-peptide complex for nuclear targeting will be presented, where three different peptide sequences are conjugated onto 10 nm sized spherical GNPs. Each of the peptides is used for enhancing intracellular retention, inducing nuclear delivery and stabilization. When in vitro cells were irradiated with 2 Gy of radiation, the cells incubated with peptide-modified GNPs had an improved therapeutic result, not only compared to the control sample (no incubation of GNP) but to cells incubated with unmodified GNPs. This research will provide insight to a more successful NP-based platform for combining treatment modalities that could eventually lead to a more effective approach in the treatment of cancer.

Nanomedicine is an exponentially growing field, and gold nanoparticles (GNPs) in particular are extensively used in research due to their abilities as anti-cancer drug carriers for chemotherapy and as dose enhancers in radiotherapy. Most GNP research in the past involved a system where GNP localization was in the cytoplasm of the cell. However, it is predicted that therapy response can be enhanced if GNPs can be effectively targeted into the nucleus. With nuclear targeting, there is a possibility in producing additional free radicals in response to irradiation within the nucleus. This can cause more damage to the DNA of cancer cells. In this review article, we discuss the successful NP-based platforms available for nuclear targeting. In addition, we also present the possible mechanisms of nuclear targeting in detail followed by its applications in cancer therapy.

Gold nanoparticles (GNPs) are emerging as promising novel agents for cancer therapy. However, the oxygen concentration in human tumors is highly heterogeneous, and there are many regions with very low levels of oxygen (hypoxia). A majority of solid tumors contain regions with oxygen pressure values of less than 0.7% in the gas phase. The purpose of this study was to investigate NP stability, toxicity, and cellular uptake under hypoxic conditions. GNPs 50 nm in diameter were used, and the experiment was performed under 0.2% (hypoxic) and 21% (normoxic) oxygen levels using MCF-7 and HeLa cells. Hypoxic cells with prolonged exposure (eighteen hours) to hypoxia had a higher NP uptake at both 6- and 24-hour NP incubation time points. No significant toxicity was introduced by NPs under hypoxic and normoxic conditions. These findings will play a vital role in the optimization of GNP-based therapeutics in cancer treatment.

To enhance PEG uptake for radiation therapy, a peptide containing an integrin binding domain (RGD) was conjugated to PEG. Nanoparticles functionalized with both the RGD peptide and PEG had a higher uptake than NPs functionalized with PEG alone.

Abstract: The interface between nanotechnology and biology needs to be well understood for improved outcome in medical applications. In other words, we need to know the interaction of nanoparticles (unit cells in nanotechnology) with cells (unit cells in biology) for improved outcome in diagnosis, imaging and therapeutic techniques. In this chapter, recent progress in the understanding of how size, shape and surface properties of nanoparticles (NPs) affect intracellular uptake, transport and processing of NPs will be discussed. Gold NPs are used as a model system in this regard as their size, shape and surface properties can be easily tailored. These findings provide useful information to tailor nanoparticle-based devices at the single cell level for effective applications in diagnosis, therapeutics and imaging.