Although liposomes are widely used as carriers of drugs and imaging agents, they suffer from a lack of stability and the slow release of the encapsulated contents at the targeted site. Polymersomes (vesicles of amphiphilic polymers) are considerably more stable compared to liposomes; however, they also demonstrate a slow release for the encapsulated contents, limiting their efficacy as a drug-delivery tool. As a solution, we prepared and characterized echogenic polymersomes which are programmed to release the encapsulated drugs rapidly when incubated with cytosolic concentrations of glutathione. These vesicles encapsulated air bubbles inside and efficiently reflected diagnostic frequency ultrasound. Folate-targeted polymersomes showed an enhanced uptake by breast and pancreatic-cancer cells in a monolayer as well as in three-dimensional spheroid cultures. Polymersomes encapsulated with the anticancer drugs gemcitabine and doxorubicin showed significant cytotoxicity to these cells. With further improvements, these vesicles hold the promise to serve as multifunctional nanocarriers, offering a triggered release as well as diagnostic ultrasound imaging.

Although lipid nanoparticles are promising drug delivery vehicles, passive release of encapsulated contents at the target site is often slow. Herein, we report contents release from targeted, polymer coated, echogenic lipid nanoparticles in the cell cytoplasm by redox trigger and simultaneously enhanced by diagnostic frequency ultrasound. The lipid nanoparticles were polymerized on the external leaflet using a disulfide cross-linker. In the presence of cytosolic concentrations of glutathione, the lipid nanoparticles released 76% of encapsulated contents. Plasma concentrations of glutathione failed to release the encapsulated contents. Application of 3 MHz ultrasound for 2 minutes simultaneously with the reducing agent enhanced the release to 96%. Folic acid conjugated, doxorubicin loaded nanoparticles showed enhanced uptake and higher cytotoxicity in cancer cells overexpressing the folate receptor (compared to the control). With further developments, these lipid nanoparticles have the potential to be used as multimodal nanocarriers for simultaneous targeted drug delivery and ultrasound imaging.

The extracellular enzyme matrix metalloproteinase-9 (MMP-9) is overexpressed in atherosclerotic plaques and in metastatic cancers. The enzyme is responsible for rupture of the plaques and for the invasion and metastasis of a large number of cancers. The ability of ultrasonic excitation to induce thermal and mechanical effects has been used to release drugs from different carriers. However, majority of these studies were performed with low frequency ultrasound (LFUS) at kHz frequencies. Clinical usage of LFUS excitations will be limited due to harmful biological effects. Herein, we report our results on the release of encapsulated contents from substrate lipopeptide incorporated echogenic liposomes triggered by recombinant human MMP-9. The contents release was further enhanced by the application of diagnostic frequency (3 MHz) ultrasound. The echogenic liposomes were successfully imaged employing a medical ultrasound transducer (4 – 15 MHz). The conditioned cell culture media from cancer cells (secreting MMP-9) released the encapsulated dye from the liposomes (30 – 50%) and this release is also increased (50 – 80%) by applying diagnostic frequency ultrasound (3 MHz) for 3 minutes. With further developments, these liposomes have the potential to serve as multimodal carriers for triggered release and simultaneous ultrasound imaging.

Chemotherapeutic agents for treating cancers show considerable side effects, toxicity, and drug resistance. To mitigate the problems, we designed nucleus-targeted, echogenic, stimuli-responsive polymeric vesicles (polymersomes) to transport and subsequently release the encapsulated anticancer drugs within the nuclei of pancreatic cancer cells. We synthesized an alkyne-dexamethasone derivative and conjugated it to N-polyethylene glycol (PEG)-polylactic acid (PLA) copolymer employing the Cu catalyzed "Click" reaction. We prepared polymersomes from the dexamethasone-PEG-PLA conjugate along with a synthesized stimuli-responsive polymer PEG-S-S-PLA. The dexamethasone group dilates the nuclear pore complexes and transports the vesicles to the nuclei. We designed the polymersomes to release the encapsulated drugs in the presence of a high concentration of reducing agents in the nuclei of pancreatic cancer cells. We observed that the nucleus-targeted, stimuli-responsive polymersomes released 70% of encapsulated contents in the nucleus-mimicking environment in 80 min. We encapsulated the cancer stemness inhibitor BBI608 in the vesicles and observed that the BBI608 encapsulated polymersomes reduced the viability of the BxPC3 cells to 43% in three-dimensional spheroid cultures. The polymersomes were prepared following a special protocol so that they scatter ultrasound, allowing imaging by a medical ultrasound scanner. Therefore, these echogenic, targeted, stimuli-responsive, and drug-encapsulated polymersomes have the potential for trackable, targeted carrier of chemotherapeutic drugs to cancer cell nuclei.

Hypoxia in solid tumors facilitates the progression of the disease, develops resistance to chemo and radiotherapy, and contributes to relapse. Due to the lack of tumor penetration, most of the reported drug carriers are unable to reach the hypoxic niches of the solid tumors. We have developed tissue-penetrating, hypoxia-responsive echogenic polymersomes to deliver anticancer drugs to solid tumors. The polymersomes are composed of a hypoxia-responsive azobenzene conjugated and a tissue penetrating peptide functionalized polylactic acid-polyethylene glycol polymer. The drug-encapsulated, hypoxia-responsive polymersomes substantially decreased the viability of pancreatic cancer cells in spheroidal cultures. Under normoxic conditions, polymersomes were echogenic at diagnostic ultrasound frequencies but lose the echogenicity under hypoxia. In-vivo imaging studies with xenograft mouse model further confirmed the ability of the polymersomes to target, penetrate, and deliver the encapsulated contents in hypoxic pancreatic tumor tissues.