Drug Delivery

Paul S, Nahire R, Mallik S, Sarkar K 2014 “Encapsulated microbubbles and echogenic liposomes for contrast ultrasound imaging and targeted drug delivery,” Computational Mechanics, 53,413-435.

Nahire R, Halder M, Paul S, Margoum A, Ambre AH, Katti KS, Gange KN, Srivastava D K, Sarkar K, Mallik S 2014 “pH-Triggered Echogenicity and Contents Release from Liposomes,” Molecular Pharamaceutics, 11, 4059-4068.

Nahire R, Halder M, Paul S, Margoum A, Ambre AH, Katti KS, Gange KN, Srivastava D K, Sarkar K, Mallik S 2013 “Polymer Coated Echogenic Lipid Nanoparticles with dual release triggers,” Biomacromolecules, 14, 841-853.

Although lipid nanoparticles are promising drugdelivery vehicles, passive release of encapsulated contents atthe target site is often slow. Herein, we report contents releasefrom targeted, polymer-coated, echogenic lipid nanoparticles inthe cell cytoplasm by redox trigger and simultaneouslyenhanced by diagnostic frequency ultrasound. The lipidnanoparticles were polymerized on the external leaflet usinga disulfide cross-linker. In the presence of cytosolicconcentrations of glutathione, the lipid nanoparticles released76% of encapsulated contents. Plasma concentrations ofglutathione failed to releasethe encapsulated contents.Application of 3 MHz ultrasound for 2 min simultaneouslywith the reducing agent enhanced the release to 96%. Folicacid conjugated, doxorubicin-loaded nanoparticles showed enhanced uptake and higher cytotoxicity in cancer cells overexpressingthe folate receptor (compared to the control). With further developments, these lipid nanoparticles have the potential to be usedas multimodal nanocarriers for simultaneous targeted drug delivery and ultrasound imaging.

Nahire R, Hossain R, Patel R, Paul S, Ambre AH, Meghnani V, Layek B, Katti KS, Gange KN, Leclarc E, Srivastava D K, Sarkar K, Mallik S 2014 “Multifunctional polymersomes for cytosolic delivery of gemcitabine and doxorubicin to cancer cells,” Biomaterials, 35, 6482-6497.

Although liposomes are widely used as carriers of drugs and imaging agents, they suffer from a lack ofstability and the slow release of the encapsulated contents at the targeted site. Polymersomes (vesicles ofamphiphilic polymers) are considerably more stable compared to liposomes; however, they alsodemonstrate 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 torelease 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 amonolayer as well as in three-dimensional spheroid cultures. Polymersomes encapsulated with theanticancer drugs gemcitabine and doxorubicin showed significant cytotoxicity to these cells. Withfurther improvements, these vesicles hold the promise to serve as multifunctional nanocarriers, offeringa triggered release as well as diagnostic ultrasound imaging.

Kumar KN, Mallik S, Sarkar K, 2017 “Role of freeze-drying in the presence of mannitol on the echogenicity of echogenic liposomes,” Journal of the Acoustical Society of America, 142, 3670-3676.

Echogenic liposomes (ELIPs) are an excellent candidate for ultrasound activated therapeutics andimaging. Although multiple experiments have established their echogenicity, the underlying mech-anism has remained unknown. However, freeze-drying in the presence of mannitol during ELIPpreparation has proved critical to ensuring echogenicity. Here, the role of this key component in thepreparation protocol was investigated by measuring scattering from freshly prepared freeze-driedaqueous solution of mannitol—and a number of other excipients commonly used in lyophiliza-tion—directly dispersed in water without any lipids in the experiment. Mannitol, meso-erythritol,glycine, and glucose that form a highly porous crystalline phase upon freeze-drying generated bub-bles resulting in strong echoes during their dissolution. On the other hand, sucrose, trehalose, andxylitol, which become glassy while freeze-dried, did not. Freeze-dried mannitol and other crystal-line substances, if thawed before being introduced into the scattering volume, did not produce echo-genicity, as they lost their crystallinity in the thawed state. The echogenicity disappeared in adegassed environment. Higher amounts of sugar in the original aqueous solution before freeze-drying resulted in higher echogenicity because of the stronger supersaturation and crystallinity. Thebubbles created by the freeze-dried mannitol in the ELIP formulation play a critical role in makingELIPs echogenic.

Xia L, Karandish F, Kumar KN, Froberg J, Kulkarni P, Gange KN, Choi Y, Mallik S, Sarkar K 2017 “Acoustic characterization of echogenic polymersomes prepared from amphiphilic block copolymers,” Ultrasound in Medicine and Biology, 44, 447-457.

Polymersomes are a class of artificial vesicles prepared from amphiphilic polymers. Like lipid vesicles(liposomes), they too can encapsulate hydrophilic and hydrophobic drug molecules in the aqueous core and thehydrophobic bilayer respectively, but are more stable than liposomes. Although echogenic liposomes have beenwidely investigated for simultaneous ultrasound imaging and controlled drug delivery, the potential of thepolymersomes remains unexplored. We prepared two different echogenic polymersomes from the amphiphilic co-polymers polyethylene glycol–poly-DL-lactic acid (PEG-PLA) and polyethylene glycol–poly-L-lactic acid (PEG-PLLA), incorporating multiple freeze-dry cycles in the synthesis protocol to ensure their echogenicity. We investigatedacoustic behavior with potential applications in biomedical imaging. We characterized the polymeric vesicles acous-tically with three different excitation frequencies of 2.25, 5 and 10 MHz at 500 kPa. The polymersomes exhibitedstrong echogenicity at all three excitation frequencies (about 50- and 25-dB enhancements in fundamental andsubharmonic, respectively, at 5-MHz excitation from 20g/mL polymers in solution). Unlike echogenic lipo-somes, they emitted strong subharmonic responses. The scattering results indicated their potential as contrastagents, which was also confirmed by clinical ultrasound imaging.

Kulkarni P, Haldar MK, Karandish F, Confeld M, Hossain R, Borowicz P, Gange KN, Xia L, Sarkar K, Mallik S 2018 “Tissue-penetrating, hypoxia-responsive echogenic polymersomes for drug delivery to solid tumors,” Chemistry A European Journal, 24, 12490-12494.

Hypoxia in solid tumors facilitates the progres-sion 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 anti cancer drugs to solid tumors. The polymersomes are composed of a hy-poxia-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.

Karandish F, Haldar MK, Xia L, Gange KN, Feng L, You S, Choi Y, Sarkar K, Mallik S  2018 “Nucleus-targeted, echogenic polymersomes for delivering a cancer stemness inhibitor to pancreatic cancer cells,” Biomacromolecules, 19,4122-4132.

Chemotherapeutic agents for treating cancers show considerable sideeffects, toxicity, and drug resistance. To mitigate the problems, we designed nucleus-targeted, echogenic, stimuli-responsive polymeric vesicles (polymersomes) to transport andsubsequently release the encapsulated anticancer drugs within the nuclei of pancreaticcancer cells. We synthesized an alkyne-dexamethasone derivative and conjugated it to N3−polyethylene glycol (PEG)−polylactic acid (PLA) copolymer employing the Cu2+catalyzed“Click”reaction. We prepared polymersomes from the dexamethasone−PEG−PLA conjugate along with a synthesized stimuli-responsive polymer PEG−S−S−PLA. Thedexamethasone group dilates the nuclear pore complexes and transports the vesicles to thenuclei. We designed the polymersomes to release the encapsulated drugs in the presence ofa high concentration of reducing agents in the nuclei of pancreatic cancer cells. Weobserved that the nucleus-targeted, stimuli-responsive polymersomes released 70% ofencapsulated contents in the nucleus-mimicking environment in 80 min. We encapsulatedthe cancer stemness inhibitor BBI608 in the vesicles and observed that the BBI608encapsulated polymersomes reduced the viability of the BxPC3 cells to 43% in three-dimensional spheroid cultures. Thepolymersomes were prepared following a special protocol so that they scatter ultrasound, allowing imaging by a medicalultrasound scanner. Therefore, these echogenic, targeted, stimuli-responsive, and drug-encapsulated polymersomes have thepotential for trackable, targeted carrier of chemotherapeutic drugs to cancer cell nuclei.

Nahire R, Halder M, Paul S, Margoum A, Ambre AH, Katti KS, Gange KN, Srivastava D K, Sarkar K, Mallik S 2014 “pH-Triggered Echogenicity and Contents Release from Liposomes,” Molecular Pharamaceutics, 11, 4059-4068.

Paul S, Nahire R, Mallik S, Sarkar K 2014 “Encapsulated microbubbles and echogenic liposomes for contrast ultrasound imaging and targeted drug delivery,” Computational Mechanics, 53,413-435.

Nahire R, Halder M, Paul S, Margoum A, Ambre AH, Katti KS, Gange KN, Srivastava D K, Sarkar K, Mallik S 2013 “Polymer Coated Echogenic Lipid Nanoparticles with dual release triggers,” Biomacromolecules, 14, 841-853.

Although lipid nanoparticles are promising drugdelivery vehicles, passive release of encapsulated contents atthe target site is often slow. Herein, we report contents releasefrom targeted, polymer-coated, echogenic lipid nanoparticles inthe cell cytoplasm by redox trigger and simultaneouslyenhanced by diagnostic frequency ultrasound. The lipidnanoparticles were polymerized on the external leaflet usinga disulfide cross-linker. In the presence of cytosolicconcentrations of glutathione, the lipid nanoparticles released76% of encapsulated contents. Plasma concentrations ofglutathione failed to releasethe encapsulated contents.Application of 3 MHz ultrasound for 2 min simultaneouslywith the reducing agent enhanced the release to 96%. Folicacid conjugated, doxorubicin-loaded nanoparticles showed enhanced uptake and higher cytotoxicity in cancer cells overexpressingthe folate receptor (compared to the control). With further developments, these lipid nanoparticles have the potential to be usedas multimodal nanocarriers for simultaneous targeted drug delivery and ultrasound imaging.

Nahire R, Hossain R, Patel R, Paul S, Ambre AH, Meghnani V, Layek B, Katti KS, Gange KN, Leclarc E, Srivastava D K, Sarkar K, Mallik S 2014 “Multifunctional polymersomes for cytosolic delivery of gemcitabine and doxorubicin to cancer cells,” Biomaterials, 35, 6482-6497.

Although liposomes are widely used as carriers of drugs and imaging agents, they suffer from a lack ofstability and the slow release of the encapsulated contents at the targeted site. Polymersomes (vesicles ofamphiphilic polymers) are considerably more stable compared to liposomes; however, they alsodemonstrate 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 torelease 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 amonolayer as well as in three-dimensional spheroid cultures. Polymersomes encapsulated with theanticancer drugs gemcitabine and doxorubicin showed significant cytotoxicity to these cells. Withfurther improvements, these vesicles hold the promise to serve as multifunctional nanocarriers, offeringa triggered release as well as diagnostic ultrasound imaging.

Kumar KN, Mallik S, Sarkar K, 2017 “Role of freeze-drying in the presence of mannitol on the echogenicity of echogenic liposomes,” Journal of the Acoustical Society of America, 142, 3670-3676.

Echogenic liposomes (ELIPs) are an excellent candidate for ultrasound activated therapeutics andimaging. Although multiple experiments have established their echogenicity, the underlying mech-anism has remained unknown. However, freeze-drying in the presence of mannitol during ELIPpreparation has proved critical to ensuring echogenicity. Here, the role of this key component in thepreparation protocol was investigated by measuring scattering from freshly prepared freeze-driedaqueous solution of mannitol—and a number of other excipients commonly used in lyophiliza-tion—directly dispersed in water without any lipids in the experiment. Mannitol, meso-erythritol,glycine, and glucose that form a highly porous crystalline phase upon freeze-drying generated bub-bles resulting in strong echoes during their dissolution. On the other hand, sucrose, trehalose, andxylitol, which become glassy while freeze-dried, did not. Freeze-dried mannitol and other crystal-line substances, if thawed before being introduced into the scattering volume, did not produce echo-genicity, as they lost their crystallinity in the thawed state. The echogenicity disappeared in adegassed environment. Higher amounts of sugar in the original aqueous solution before freeze-drying resulted in higher echogenicity because of the stronger supersaturation and crystallinity. Thebubbles created by the freeze-dried mannitol in the ELIP formulation play a critical role in makingELIPs echogenic.

Xia L, Karandish F, Kumar KN, Froberg J, Kulkarni P, Gange KN, Choi Y, Mallik S, Sarkar K 2017 “Acoustic characterization of echogenic polymersomes prepared from amphiphilic block copolymers,” Ultrasound in Medicine and Biology, 44, 447-457.

Polymersomes are a class of artificial vesicles prepared from amphiphilic polymers. Like lipid vesicles(liposomes), they too can encapsulate hydrophilic and hydrophobic drug molecules in the aqueous core and thehydrophobic bilayer respectively, but are more stable than liposomes. Although echogenic liposomes have beenwidely investigated for simultaneous ultrasound imaging and controlled drug delivery, the potential of thepolymersomes remains unexplored. We prepared two different echogenic polymersomes from the amphiphilic co-polymers polyethylene glycol–poly-DL-lactic acid (PEG-PLA) and polyethylene glycol–poly-L-lactic acid (PEG-PLLA), incorporating multiple freeze-dry cycles in the synthesis protocol to ensure their echogenicity. We investigatedacoustic behavior with potential applications in biomedical imaging. We characterized the polymeric vesicles acous-tically with three different excitation frequencies of 2.25, 5 and 10 MHz at 500 kPa. The polymersomes exhibitedstrong echogenicity at all three excitation frequencies (about 50- and 25-dB enhancements in fundamental andsubharmonic, respectively, at 5-MHz excitation from 20g/mL polymers in solution). Unlike echogenic lipo-somes, they emitted strong subharmonic responses. The scattering results indicated their potential as contrastagents, which was also confirmed by clinical ultrasound imaging.

Kulkarni P, Haldar MK, Karandish F, Confeld M, Hossain R, Borowicz P, Gange KN, Xia L, Sarkar K, Mallik S 2018 “Tissue-penetrating, hypoxia-responsive echogenic polymersomes for drug delivery to solid tumors,” Chemistry A European Journal, 24, 12490-12494.

Hypoxia in solid tumors facilitates the progres-sion 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 anti cancer drugs to solid tumors. The polymersomes are composed of a hy-poxia-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.

Karandish F, Haldar MK, Xia L, Gange KN, Feng L, You S, Choi Y, Sarkar K, Mallik S  2018 “Nucleus-targeted, echogenic polymersomes for delivering a cancer stemness inhibitor to pancreatic cancer cells,” Biomacromolecules, 19,4122-4132.

Chemotherapeutic agents for treating cancers show considerable sideeffects, toxicity, and drug resistance. To mitigate the problems, we designed nucleus-targeted, echogenic, stimuli-responsive polymeric vesicles (polymersomes) to transport andsubsequently release the encapsulated anticancer drugs within the nuclei of pancreaticcancer cells. We synthesized an alkyne-dexamethasone derivative and conjugated it to N3−polyethylene glycol (PEG)−polylactic acid (PLA) copolymer employing the Cu2+catalyzed“Click”reaction. We prepared polymersomes from the dexamethasone−PEG−PLA conjugate along with a synthesized stimuli-responsive polymer PEG−S−S−PLA. Thedexamethasone group dilates the nuclear pore complexes and transports the vesicles to thenuclei. We designed the polymersomes to release the encapsulated drugs in the presence ofa high concentration of reducing agents in the nuclei of pancreatic cancer cells. Weobserved that the nucleus-targeted, stimuli-responsive polymersomes released 70% ofencapsulated contents in the nucleus-mimicking environment in 80 min. We encapsulatedthe cancer stemness inhibitor BBI608 in the vesicles and observed that the BBI608encapsulated polymersomes reduced the viability of the BxPC3 cells to 43% in three-dimensional spheroid cultures. Thepolymersomes were prepared following a special protocol so that they scatter ultrasound, allowing imaging by a medicalultrasound scanner. Therefore, these echogenic, targeted, stimuli-responsive, and drug-encapsulated polymersomes have thepotential for trackable, targeted carrier of chemotherapeutic drugs to cancer cell nuclei.

Karandish F, Haldar MK, Xia L, Gange KN, Feng L, You S, Choi Y, Sarkar K, Mallik S  2018 “Nucleus-targeted, echogenic polymersomes for delivering a cancer stemness inhibitor to pancreatic cancer cells,” Biomacromolecules, 19,4122-4132.

Chemotherapeutic agents for treating cancers show considerable sideeffects, toxicity, and drug resistance. To mitigate the problems, we designed nucleus-targeted, echogenic, stimuli-responsive polymeric vesicles (polymersomes) to transport andsubsequently release the encapsulated anticancer drugs within the nuclei of pancreaticcancer cells. We synthesized an alkyne-dexamethasone derivative and conjugated it to N3−polyethylene glycol (PEG)−polylactic acid (PLA) copolymer employing the Cu2+catalyzed“Click”reaction. We prepared polymersomes from the dexamethasone−PEG−PLA conjugate along with a synthesized stimuli-responsive polymer PEG−S−S−PLA. Thedexamethasone group dilates the nuclear pore complexes and transports the vesicles to thenuclei. We designed the polymersomes to release the encapsulated drugs in the presence ofa high concentration of reducing agents in the nuclei of pancreatic cancer cells. Weobserved that the nucleus-targeted, stimuli-responsive polymersomes released 70% ofencapsulated contents in the nucleus-mimicking environment in 80 min. We encapsulatedthe cancer stemness inhibitor BBI608 in the vesicles and observed that the BBI608encapsulated polymersomes reduced the viability of the BxPC3 cells to 43% in three-dimensional spheroid cultures. Thepolymersomes were prepared following a special protocol so that they scatter ultrasound, allowing imaging by a medicalultrasound scanner. Therefore, these echogenic, targeted, stimuli-responsive, and drug-encapsulated polymersomes have thepotential for trackable, targeted carrier of chemotherapeutic drugs to cancer cell nuclei.

Kulkarni P, Haldar MK, Karandish F, Confeld M, Hossain R, Borowicz P, Gange KN, Xia L, Sarkar K, Mallik S 2018 “Tissue-penetrating, hypoxia-responsive echogenic polymersomes for drug delivery to solid tumors,” Chemistry A European Journal, 24, 12490-12494.

Hypoxia in solid tumors facilitates the progres-sion 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 anti cancer drugs to solid tumors. The polymersomes are composed of a hy-poxia-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.

Kulkarni P, Haldar MK, Karandish F, Confeld M, Hossain R, Borowicz P, Gange KN, Xia L, Sarkar K, Mallik S 2018 “Tissue-penetrating, hypoxia-responsive echogenic polymersomes for drug delivery to solid tumors,” Chemistry A European Journal, 24, 12490-12494.

Hypoxia in solid tumors facilitates the progres-sion 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 anti cancer drugs to solid tumors. The polymersomes are composed of a hy-poxia-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.

Kumar KN, Mallik S, Sarkar K, 2017 “Role of freeze-drying in the presence of mannitol on the echogenicity of echogenic liposomes,” Journal of the Acoustical Society of America, 142, 3670-3676.

Echogenic liposomes (ELIPs) are an excellent candidate for ultrasound activated therapeutics andimaging. Although multiple experiments have established their echogenicity, the underlying mech-anism has remained unknown. However, freeze-drying in the presence of mannitol during ELIPpreparation has proved critical to ensuring echogenicity. Here, the role of this key component in thepreparation protocol was investigated by measuring scattering from freshly prepared freeze-driedaqueous solution of mannitol—and a number of other excipients commonly used in lyophiliza-tion—directly dispersed in water without any lipids in the experiment. Mannitol, meso-erythritol,glycine, and glucose that form a highly porous crystalline phase upon freeze-drying generated bub-bles resulting in strong echoes during their dissolution. On the other hand, sucrose, trehalose, andxylitol, which become glassy while freeze-dried, did not. Freeze-dried mannitol and other crystal-line substances, if thawed before being introduced into the scattering volume, did not produce echo-genicity, as they lost their crystallinity in the thawed state. The echogenicity disappeared in adegassed environment. Higher amounts of sugar in the original aqueous solution before freeze-drying resulted in higher echogenicity because of the stronger supersaturation and crystallinity. Thebubbles created by the freeze-dried mannitol in the ELIP formulation play a critical role in makingELIPs echogenic.

Xia L, Karandish F, Kumar KN, Froberg J, Kulkarni P, Gange KN, Choi Y, Mallik S, Sarkar K 2017 “Acoustic characterization of echogenic polymersomes prepared from amphiphilic block copolymers,” Ultrasound in Medicine and Biology, 44, 447-457.

Polymersomes are a class of artificial vesicles prepared from amphiphilic polymers. Like lipid vesicles(liposomes), they too can encapsulate hydrophilic and hydrophobic drug molecules in the aqueous core and thehydrophobic bilayer respectively, but are more stable than liposomes. Although echogenic liposomes have beenwidely investigated for simultaneous ultrasound imaging and controlled drug delivery, the potential of thepolymersomes remains unexplored. We prepared two different echogenic polymersomes from the amphiphilic co-polymers polyethylene glycol–poly-DL-lactic acid (PEG-PLA) and polyethylene glycol–poly-L-lactic acid (PEG-PLLA), incorporating multiple freeze-dry cycles in the synthesis protocol to ensure their echogenicity. We investigatedacoustic behavior with potential applications in biomedical imaging. We characterized the polymeric vesicles acous-tically with three different excitation frequencies of 2.25, 5 and 10 MHz at 500 kPa. The polymersomes exhibitedstrong echogenicity at all three excitation frequencies (about 50- and 25-dB enhancements in fundamental andsubharmonic, respectively, at 5-MHz excitation from 20g/mL polymers in solution). Unlike echogenic lipo-somes, they emitted strong subharmonic responses. The scattering results indicated their potential as contrastagents, which was also confirmed by clinical ultrasound imaging.

Kulkarni P, Haldar MK, Karandish F, Confeld M, Hossain R, Borowicz P, Gange KN, Xia L, Sarkar K, Mallik S 2018 “Tissue-penetrating, hypoxia-responsive echogenic polymersomes for drug delivery to solid tumors,” Chemistry A European Journal, 24, 12490-12494.

Hypoxia in solid tumors facilitates the progres-sion 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 anti cancer drugs to solid tumors. The polymersomes are composed of a hy-poxia-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.

Nahire R, Halder M, Paul S, Margoum A, Ambre AH, Katti KS, Gange KN, Srivastava D K, Sarkar K, Mallik S 2014 “pH-Triggered Echogenicity and Contents Release from Liposomes,” Molecular Pharamaceutics, 11, 4059-4068.

Nahire R, Hossain R, Patel R, Paul S, Ambre AH, Meghnani V, Layek B, Katti KS, Gange KN, Leclarc E, Srivastava D K, Sarkar K, Mallik S 2014 “Multifunctional polymersomes for cytosolic delivery of gemcitabine and doxorubicin to cancer cells,” Biomaterials, 35, 6482-6497.

Although liposomes are widely used as carriers of drugs and imaging agents, they suffer from a lack ofstability and the slow release of the encapsulated contents at the targeted site. Polymersomes (vesicles ofamphiphilic polymers) are considerably more stable compared to liposomes; however, they alsodemonstrate 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 torelease 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 amonolayer as well as in three-dimensional spheroid cultures. Polymersomes encapsulated with theanticancer drugs gemcitabine and doxorubicin showed significant cytotoxicity to these cells. Withfurther improvements, these vesicles hold the promise to serve as multifunctional nanocarriers, offeringa triggered release as well as diagnostic ultrasound imaging.

Paul S, Nahire R, Mallik S, Sarkar K 2014 “Encapsulated microbubbles and echogenic liposomes for contrast ultrasound imaging and targeted drug delivery,” Computational Mechanics, 53,413-435.

Nahire R, Halder M, Paul S, Margoum A, Ambre AH, Katti KS, Gange KN, Srivastava D K, Sarkar K, Mallik S 2013 “Polymer Coated Echogenic Lipid Nanoparticles with dual release triggers,” Biomacromolecules, 14, 841-853.

Although lipid nanoparticles are promising drugdelivery vehicles, passive release of encapsulated contents atthe target site is often slow. Herein, we report contents releasefrom targeted, polymer-coated, echogenic lipid nanoparticles inthe cell cytoplasm by redox trigger and simultaneouslyenhanced by diagnostic frequency ultrasound. The lipidnanoparticles were polymerized on the external leaflet usinga disulfide cross-linker. In the presence of cytosolicconcentrations of glutathione, the lipid nanoparticles released76% of encapsulated contents. Plasma concentrations ofglutathione failed to releasethe encapsulated contents.Application of 3 MHz ultrasound for 2 min simultaneouslywith the reducing agent enhanced the release to 96%. Folicacid conjugated, doxorubicin-loaded nanoparticles showed enhanced uptake and higher cytotoxicity in cancer cells overexpressingthe folate receptor (compared to the control). With further developments, these lipid nanoparticles have the potential to be usedas multimodal nanocarriers for simultaneous targeted drug delivery and ultrasound imaging.

Kulkarni P, Haldar MK, Karandish F, Confeld M, Hossain R, Borowicz P, Gange KN, Xia L, Sarkar K, Mallik S 2018 “Tissue-penetrating, hypoxia-responsive echogenic polymersomes for drug delivery to solid tumors,” Chemistry A European Journal, 24, 12490-12494.

Hypoxia in solid tumors facilitates the progres-sion 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 anti cancer drugs to solid tumors. The polymersomes are composed of a hy-poxia-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.

Karandish F, Haldar MK, Xia L, Gange KN, Feng L, You S, Choi Y, Sarkar K, Mallik S  2018 “Nucleus-targeted, echogenic polymersomes for delivering a cancer stemness inhibitor to pancreatic cancer cells,” Biomacromolecules, 19,4122-4132.

Chemotherapeutic agents for treating cancers show considerable sideeffects, toxicity, and drug resistance. To mitigate the problems, we designed nucleus-targeted, echogenic, stimuli-responsive polymeric vesicles (polymersomes) to transport andsubsequently release the encapsulated anticancer drugs within the nuclei of pancreaticcancer cells. We synthesized an alkyne-dexamethasone derivative and conjugated it to N3−polyethylene glycol (PEG)−polylactic acid (PLA) copolymer employing the Cu2+catalyzed“Click”reaction. We prepared polymersomes from the dexamethasone−PEG−PLA conjugate along with a synthesized stimuli-responsive polymer PEG−S−S−PLA. Thedexamethasone group dilates the nuclear pore complexes and transports the vesicles to thenuclei. We designed the polymersomes to release the encapsulated drugs in the presence ofa high concentration of reducing agents in the nuclei of pancreatic cancer cells. Weobserved that the nucleus-targeted, stimuli-responsive polymersomes released 70% ofencapsulated contents in the nucleus-mimicking environment in 80 min. We encapsulatedthe cancer stemness inhibitor BBI608 in the vesicles and observed that the BBI608encapsulated polymersomes reduced the viability of the BxPC3 cells to 43% in three-dimensional spheroid cultures. Thepolymersomes were prepared following a special protocol so that they scatter ultrasound, allowing imaging by a medicalultrasound scanner. Therefore, these echogenic, targeted, stimuli-responsive, and drug-encapsulated polymersomes have thepotential for trackable, targeted carrier of chemotherapeutic drugs to cancer cell nuclei.

Kulkarni P, Haldar MK, Karandish F, Confeld M, Hossain R, Borowicz P, Gange KN, Xia L, Sarkar K, Mallik S 2018 “Tissue-penetrating, hypoxia-responsive echogenic polymersomes for drug delivery to solid tumors,” Chemistry A European Journal, 24, 12490-12494.

Hypoxia in solid tumors facilitates the progres-sion 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 anti cancer drugs to solid tumors. The polymersomes are composed of a hy-poxia-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.

Karandish F, Haldar MK, Xia L, Gange KN, Feng L, You S, Choi Y, Sarkar K, Mallik S  2018 “Nucleus-targeted, echogenic polymersomes for delivering a cancer stemness inhibitor to pancreatic cancer cells,” Biomacromolecules, 19,4122-4132.

Chemotherapeutic agents for treating cancers show considerable sideeffects, toxicity, and drug resistance. To mitigate the problems, we designed nucleus-targeted, echogenic, stimuli-responsive polymeric vesicles (polymersomes) to transport andsubsequently release the encapsulated anticancer drugs within the nuclei of pancreaticcancer cells. We synthesized an alkyne-dexamethasone derivative and conjugated it to N3−polyethylene glycol (PEG)−polylactic acid (PLA) copolymer employing the Cu2+catalyzed“Click”reaction. We prepared polymersomes from the dexamethasone−PEG−PLA conjugate along with a synthesized stimuli-responsive polymer PEG−S−S−PLA. Thedexamethasone group dilates the nuclear pore complexes and transports the vesicles to thenuclei. We designed the polymersomes to release the encapsulated drugs in the presence ofa high concentration of reducing agents in the nuclei of pancreatic cancer cells. Weobserved that the nucleus-targeted, stimuli-responsive polymersomes released 70% ofencapsulated contents in the nucleus-mimicking environment in 80 min. We encapsulatedthe cancer stemness inhibitor BBI608 in the vesicles and observed that the BBI608encapsulated polymersomes reduced the viability of the BxPC3 cells to 43% in three-dimensional spheroid cultures. Thepolymersomes were prepared following a special protocol so that they scatter ultrasound, allowing imaging by a medicalultrasound scanner. Therefore, these echogenic, targeted, stimuli-responsive, and drug-encapsulated polymersomes have thepotential for trackable, targeted carrier of chemotherapeutic drugs to cancer cell nuclei.

Kumar KN, Mallik S, Sarkar K, 2017 “Role of freeze-drying in the presence of mannitol on the echogenicity of echogenic liposomes,” Journal of the Acoustical Society of America, 142, 3670-3676.

Echogenic liposomes (ELIPs) are an excellent candidate for ultrasound activated therapeutics andimaging. Although multiple experiments have established their echogenicity, the underlying mech-anism has remained unknown. However, freeze-drying in the presence of mannitol during ELIPpreparation has proved critical to ensuring echogenicity. Here, the role of this key component in thepreparation protocol was investigated by measuring scattering from freshly prepared freeze-driedaqueous solution of mannitol—and a number of other excipients commonly used in lyophiliza-tion—directly dispersed in water without any lipids in the experiment. Mannitol, meso-erythritol,glycine, and glucose that form a highly porous crystalline phase upon freeze-drying generated bub-bles resulting in strong echoes during their dissolution. On the other hand, sucrose, trehalose, andxylitol, which become glassy while freeze-dried, did not. Freeze-dried mannitol and other crystal-line substances, if thawed before being introduced into the scattering volume, did not produce echo-genicity, as they lost their crystallinity in the thawed state. The echogenicity disappeared in adegassed environment. Higher amounts of sugar in the original aqueous solution before freeze-drying resulted in higher echogenicity because of the stronger supersaturation and crystallinity. Thebubbles created by the freeze-dried mannitol in the ELIP formulation play a critical role in makingELIPs echogenic.

Xia L, Karandish F, Kumar KN, Froberg J, Kulkarni P, Gange KN, Choi Y, Mallik S, Sarkar K 2017 “Acoustic characterization of echogenic polymersomes prepared from amphiphilic block copolymers,” Ultrasound in Medicine and Biology, 44, 447-457.

Polymersomes are a class of artificial vesicles prepared from amphiphilic polymers. Like lipid vesicles(liposomes), they too can encapsulate hydrophilic and hydrophobic drug molecules in the aqueous core and thehydrophobic bilayer respectively, but are more stable than liposomes. Although echogenic liposomes have beenwidely investigated for simultaneous ultrasound imaging and controlled drug delivery, the potential of thepolymersomes remains unexplored. We prepared two different echogenic polymersomes from the amphiphilic co-polymers polyethylene glycol–poly-DL-lactic acid (PEG-PLA) and polyethylene glycol–poly-L-lactic acid (PEG-PLLA), incorporating multiple freeze-dry cycles in the synthesis protocol to ensure their echogenicity. We investigatedacoustic behavior with potential applications in biomedical imaging. We characterized the polymeric vesicles acous-tically with three different excitation frequencies of 2.25, 5 and 10 MHz at 500 kPa. The polymersomes exhibitedstrong echogenicity at all three excitation frequencies (about 50- and 25-dB enhancements in fundamental andsubharmonic, respectively, at 5-MHz excitation from 20g/mL polymers in solution). Unlike echogenic lipo-somes, they emitted strong subharmonic responses. The scattering results indicated their potential as contrastagents, which was also confirmed by clinical ultrasound imaging.

Nahire R, Halder M, Paul S, Margoum A, Ambre AH, Katti KS, Gange KN, Srivastava D K, Sarkar K, Mallik S 2014 “pH-Triggered Echogenicity and Contents Release from Liposomes,” Molecular Pharamaceutics, 11, 4059-4068.

Paul S, Nahire R, Mallik S, Sarkar K 2014 “Encapsulated microbubbles and echogenic liposomes for contrast ultrasound imaging and targeted drug delivery,” Computational Mechanics, 53,413-435.

Nahire R, Hossain R, Patel R, Paul S, Ambre AH, Meghnani V, Layek B, Katti KS, Gange KN, Leclarc E, Srivastava D K, Sarkar K, Mallik S 2014 “Multifunctional polymersomes for cytosolic delivery of gemcitabine and doxorubicin to cancer cells,” Biomaterials, 35, 6482-6497.

Although liposomes are widely used as carriers of drugs and imaging agents, they suffer from a lack ofstability and the slow release of the encapsulated contents at the targeted site. Polymersomes (vesicles ofamphiphilic polymers) are considerably more stable compared to liposomes; however, they alsodemonstrate 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 torelease 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 amonolayer as well as in three-dimensional spheroid cultures. Polymersomes encapsulated with theanticancer drugs gemcitabine and doxorubicin showed significant cytotoxicity to these cells. Withfurther improvements, these vesicles hold the promise to serve as multifunctional nanocarriers, offeringa triggered release as well as diagnostic ultrasound imaging.

Karandish F, Haldar MK, Xia L, Gange KN, Feng L, You S, Choi Y, Sarkar K, Mallik S  2018 “Nucleus-targeted, echogenic polymersomes for delivering a cancer stemness inhibitor to pancreatic cancer cells,” Biomacromolecules, 19,4122-4132.

Chemotherapeutic agents for treating cancers show considerable sideeffects, toxicity, and drug resistance. To mitigate the problems, we designed nucleus-targeted, echogenic, stimuli-responsive polymeric vesicles (polymersomes) to transport andsubsequently release the encapsulated anticancer drugs within the nuclei of pancreaticcancer cells. We synthesized an alkyne-dexamethasone derivative and conjugated it to N3−polyethylene glycol (PEG)−polylactic acid (PLA) copolymer employing the Cu2+catalyzed“Click”reaction. We prepared polymersomes from the dexamethasone−PEG−PLA conjugate along with a synthesized stimuli-responsive polymer PEG−S−S−PLA. Thedexamethasone group dilates the nuclear pore complexes and transports the vesicles to thenuclei. We designed the polymersomes to release the encapsulated drugs in the presence ofa high concentration of reducing agents in the nuclei of pancreatic cancer cells. Weobserved that the nucleus-targeted, stimuli-responsive polymersomes released 70% ofencapsulated contents in the nucleus-mimicking environment in 80 min. We encapsulatedthe cancer stemness inhibitor BBI608 in the vesicles and observed that the BBI608encapsulated polymersomes reduced the viability of the BxPC3 cells to 43% in three-dimensional spheroid cultures. Thepolymersomes were prepared following a special protocol so that they scatter ultrasound, allowing imaging by a medicalultrasound scanner. Therefore, these echogenic, targeted, stimuli-responsive, and drug-encapsulated polymersomes have thepotential for trackable, targeted carrier of chemotherapeutic drugs to cancer cell nuclei.

Nahire R, Halder M, Paul S, Margoum A, Ambre AH, Katti KS, Gange KN, Srivastava D K, Sarkar K, Mallik S 2013 “Polymer Coated Echogenic Lipid Nanoparticles with dual release triggers,” Biomacromolecules, 14, 841-853.

Although lipid nanoparticles are promising drugdelivery vehicles, passive release of encapsulated contents atthe target site is often slow. Herein, we report contents releasefrom targeted, polymer-coated, echogenic lipid nanoparticles inthe cell cytoplasm by redox trigger and simultaneouslyenhanced by diagnostic frequency ultrasound. The lipidnanoparticles were polymerized on the external leaflet usinga disulfide cross-linker. In the presence of cytosolicconcentrations of glutathione, the lipid nanoparticles released76% of encapsulated contents. Plasma concentrations ofglutathione failed to releasethe encapsulated contents.Application of 3 MHz ultrasound for 2 min simultaneouslywith the reducing agent enhanced the release to 96%. Folicacid conjugated, doxorubicin-loaded nanoparticles showed enhanced uptake and higher cytotoxicity in cancer cells overexpressingthe folate receptor (compared to the control). With further developments, these lipid nanoparticles have the potential to be usedas multimodal nanocarriers for simultaneous targeted drug delivery and ultrasound imaging.

Pullan J, Confeld M, Osborn J,  Kim J, Sarkar K,  Mallik S, 2019 “Exosomes as drug carriers for cancer therapy,” Molecular Pharamaceutics, 16, 1789-1798.

Exosomes, biological extracellular vesicles, have recently begun to find use in targeted drug delivery in solid tumor research. Ranging from 30−120 nm in size, exosomes are secreted from cells and
isolated from bodily fluids. Exosomes provide a unique material platform due to their characteristics, including physical properties such as stability, biocompatibility, permeability, low toxicity, and low immunogenicity—all critical to the success of any nanoparticle drug
delivery system. In addition to traditional chemotherapeutics, natural products and RNA have been encapsulated for the treatment of breast, pancreatic, lung, prostate cancers, and glioblastoma. This review discusses current research on exosomes for drug delivery to solid
tumors.

Pullan J, Confeld M, Osborn J,  Kim J, Sarkar K,  Mallik S, 2019 “Exosomes as drug carriers for cancer therapy,” Molecular Pharamaceutics, 16, 1789-1798.

Exosomes, biological extracellular vesicles, have recently begun to find use in targeted drug delivery in solid tumor research. Ranging from 30−120 nm in size, exosomes are secreted from cells and
isolated from bodily fluids. Exosomes provide a unique material platform due to their characteristics, including physical properties such as stability, biocompatibility, permeability, low toxicity, and low immunogenicity—all critical to the success of any nanoparticle drug
delivery system. In addition to traditional chemotherapeutics, natural products and RNA have been encapsulated for the treatment of breast, pancreatic, lung, prostate cancers, and glioblastoma. This review discusses current research on exosomes for drug delivery to solid
tumors.

Pullan J, Confeld M, Osborn J,  Kim J, Sarkar K,  Mallik S, 2019 “Exosomes as drug carriers for cancer therapy,” Molecular Pharamaceutics, 16, 1789-1798.

Exosomes, biological extracellular vesicles, have recently begun to find use in targeted drug delivery in solid tumor research. Ranging from 30−120 nm in size, exosomes are secreted from cells and
isolated from bodily fluids. Exosomes provide a unique material platform due to their characteristics, including physical properties such as stability, biocompatibility, permeability, low toxicity, and low immunogenicity—all critical to the success of any nanoparticle drug
delivery system. In addition to traditional chemotherapeutics, natural products and RNA have been encapsulated for the treatment of breast, pancreatic, lung, prostate cancers, and glioblastoma. This review discusses current research on exosomes for drug delivery to solid
tumors.

Kulkarni P, Haldar MK, Karandish F, Confeld M, Hossain R, Borowicz P, Gange KN, Xia L, Sarkar K, Mallik S 2018 “Tissue-penetrating, hypoxia-responsive echogenic polymersomes for drug delivery to solid tumors,” Chemistry A European Journal, 24, 12490-12494.

Hypoxia in solid tumors facilitates the progres-sion 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 anti cancer drugs to solid tumors. The polymersomes are composed of a hy-poxia-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.

Karandish F, Haldar MK, Xia L, Gange KN, Feng L, You S, Choi Y, Sarkar K, Mallik S  2018 “Nucleus-targeted, echogenic polymersomes for delivering a cancer stemness inhibitor to pancreatic cancer cells,” Biomacromolecules, 19,4122-4132.

Chemotherapeutic agents for treating cancers show considerable sideeffects, toxicity, and drug resistance. To mitigate the problems, we designed nucleus-targeted, echogenic, stimuli-responsive polymeric vesicles (polymersomes) to transport andsubsequently release the encapsulated anticancer drugs within the nuclei of pancreaticcancer cells. We synthesized an alkyne-dexamethasone derivative and conjugated it to N3−polyethylene glycol (PEG)−polylactic acid (PLA) copolymer employing the Cu2+catalyzed“Click”reaction. We prepared polymersomes from the dexamethasone−PEG−PLA conjugate along with a synthesized stimuli-responsive polymer PEG−S−S−PLA. Thedexamethasone group dilates the nuclear pore complexes and transports the vesicles to thenuclei. We designed the polymersomes to release the encapsulated drugs in the presence ofa high concentration of reducing agents in the nuclei of pancreatic cancer cells. Weobserved that the nucleus-targeted, stimuli-responsive polymersomes released 70% ofencapsulated contents in the nucleus-mimicking environment in 80 min. We encapsulatedthe cancer stemness inhibitor BBI608 in the vesicles and observed that the BBI608encapsulated polymersomes reduced the viability of the BxPC3 cells to 43% in three-dimensional spheroid cultures. Thepolymersomes were prepared following a special protocol so that they scatter ultrasound, allowing imaging by a medicalultrasound scanner. Therefore, these echogenic, targeted, stimuli-responsive, and drug-encapsulated polymersomes have thepotential for trackable, targeted carrier of chemotherapeutic drugs to cancer cell nuclei.

Xia L, Karandish F, Kumar KN, Froberg J, Kulkarni P, Gange KN, Choi Y, Mallik S, Sarkar K 2017 “Acoustic characterization of echogenic polymersomes prepared from amphiphilic block copolymers,” Ultrasound in Medicine and Biology, 44, 447-457.

Polymersomes are a class of artificial vesicles prepared from amphiphilic polymers. Like lipid vesicles(liposomes), they too can encapsulate hydrophilic and hydrophobic drug molecules in the aqueous core and thehydrophobic bilayer respectively, but are more stable than liposomes. Although echogenic liposomes have beenwidely investigated for simultaneous ultrasound imaging and controlled drug delivery, the potential of thepolymersomes remains unexplored. We prepared two different echogenic polymersomes from the amphiphilic co-polymers polyethylene glycol–poly-DL-lactic acid (PEG-PLA) and polyethylene glycol–poly-L-lactic acid (PEG-PLLA), incorporating multiple freeze-dry cycles in the synthesis protocol to ensure their echogenicity. We investigatedacoustic behavior with potential applications in biomedical imaging. We characterized the polymeric vesicles acous-tically with three different excitation frequencies of 2.25, 5 and 10 MHz at 500 kPa. The polymersomes exhibitedstrong echogenicity at all three excitation frequencies (about 50- and 25-dB enhancements in fundamental andsubharmonic, respectively, at 5-MHz excitation from 20g/mL polymers in solution). Unlike echogenic lipo-somes, they emitted strong subharmonic responses. The scattering results indicated their potential as contrastagents, which was also confirmed by clinical ultrasound imaging.

Kumar KN, Mallik S, Sarkar K, 2017 “Role of freeze-drying in the presence of mannitol on the echogenicity of echogenic liposomes,” Journal of the Acoustical Society of America, 142, 3670-3676.

Echogenic liposomes (ELIPs) are an excellent candidate for ultrasound activated therapeutics andimaging. Although multiple experiments have established their echogenicity, the underlying mech-anism has remained unknown. However, freeze-drying in the presence of mannitol during ELIPpreparation has proved critical to ensuring echogenicity. Here, the role of this key component in thepreparation protocol was investigated by measuring scattering from freshly prepared freeze-driedaqueous solution of mannitol—and a number of other excipients commonly used in lyophiliza-tion—directly dispersed in water without any lipids in the experiment. Mannitol, meso-erythritol,glycine, and glucose that form a highly porous crystalline phase upon freeze-drying generated bub-bles resulting in strong echoes during their dissolution. On the other hand, sucrose, trehalose, andxylitol, which become glassy while freeze-dried, did not. Freeze-dried mannitol and other crystal-line substances, if thawed before being introduced into the scattering volume, did not produce echo-genicity, as they lost their crystallinity in the thawed state. The echogenicity disappeared in adegassed environment. Higher amounts of sugar in the original aqueous solution before freeze-drying resulted in higher echogenicity because of the stronger supersaturation and crystallinity. Thebubbles created by the freeze-dried mannitol in the ELIP formulation play a critical role in makingELIPs echogenic.

Nahire R, Hossain R, Patel R, Paul S, Ambre AH, Meghnani V, Layek B, Katti KS, Gange KN, Leclarc E, Srivastava D K, Sarkar K, Mallik S 2014 “Multifunctional polymersomes for cytosolic delivery of gemcitabine and doxorubicin to cancer cells,” Biomaterials, 35, 6482-6497.

Although liposomes are widely used as carriers of drugs and imaging agents, they suffer from a lack ofstability and the slow release of the encapsulated contents at the targeted site. Polymersomes (vesicles ofamphiphilic polymers) are considerably more stable compared to liposomes; however, they alsodemonstrate 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 torelease 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 amonolayer as well as in three-dimensional spheroid cultures. Polymersomes encapsulated with theanticancer drugs gemcitabine and doxorubicin showed significant cytotoxicity to these cells. Withfurther improvements, these vesicles hold the promise to serve as multifunctional nanocarriers, offeringa triggered release as well as diagnostic ultrasound imaging.

Nahire R, Halder M, Paul S, Margoum A, Ambre AH, Katti KS, Gange KN, Srivastava D K, Sarkar K, Mallik S 2014 “pH-Triggered Echogenicity and Contents Release from Liposomes,” Molecular Pharamaceutics, 11, 4059-4068.

Paul S, Nahire R, Mallik S, Sarkar K 2014 “Encapsulated microbubbles and echogenic liposomes for contrast ultrasound imaging and targeted drug delivery,” Computational Mechanics, 53,413-435.

Nahire R, Halder M, Paul S, Margoum A, Ambre AH, Katti KS, Gange KN, Srivastava D K, Sarkar K, Mallik S 2013 “Polymer Coated Echogenic Lipid Nanoparticles with dual release triggers,” Biomacromolecules, 14, 841-853.

Although lipid nanoparticles are promising drugdelivery vehicles, passive release of encapsulated contents atthe target site is often slow. Herein, we report contents releasefrom targeted, polymer-coated, echogenic lipid nanoparticles inthe cell cytoplasm by redox trigger and simultaneouslyenhanced by diagnostic frequency ultrasound. The lipidnanoparticles were polymerized on the external leaflet usinga disulfide cross-linker. In the presence of cytosolicconcentrations of glutathione, the lipid nanoparticles released76% of encapsulated contents. Plasma concentrations ofglutathione failed to releasethe encapsulated contents.Application of 3 MHz ultrasound for 2 min simultaneouslywith the reducing agent enhanced the release to 96%. Folicacid conjugated, doxorubicin-loaded nanoparticles showed enhanced uptake and higher cytotoxicity in cancer cells overexpressingthe folate receptor (compared to the control). With further developments, these lipid nanoparticles have the potential to be usedas multimodal nanocarriers for simultaneous targeted drug delivery and ultrasound imaging.

Kulkarni P, Haldar MK, Karandish F, Confeld M, Hossain R, Borowicz P, Gange KN, Xia L, Sarkar K, Mallik S 2018 “Tissue-penetrating, hypoxia-responsive echogenic polymersomes for drug delivery to solid tumors,” Chemistry A European Journal, 24, 12490-12494.

Hypoxia in solid tumors facilitates the progres-sion 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 anti cancer drugs to solid tumors. The polymersomes are composed of a hy-poxia-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.

Karandish F, Haldar MK, Xia L, Gange KN, Feng L, You S, Choi Y, Sarkar K, Mallik S  2018 “Nucleus-targeted, echogenic polymersomes for delivering a cancer stemness inhibitor to pancreatic cancer cells,” Biomacromolecules, 19,4122-4132.

Chemotherapeutic agents for treating cancers show considerable sideeffects, toxicity, and drug resistance. To mitigate the problems, we designed nucleus-targeted, echogenic, stimuli-responsive polymeric vesicles (polymersomes) to transport andsubsequently release the encapsulated anticancer drugs within the nuclei of pancreaticcancer cells. We synthesized an alkyne-dexamethasone derivative and conjugated it to N3−polyethylene glycol (PEG)−polylactic acid (PLA) copolymer employing the Cu2+catalyzed“Click”reaction. We prepared polymersomes from the dexamethasone−PEG−PLA conjugate along with a synthesized stimuli-responsive polymer PEG−S−S−PLA. Thedexamethasone group dilates the nuclear pore complexes and transports the vesicles to thenuclei. We designed the polymersomes to release the encapsulated drugs in the presence ofa high concentration of reducing agents in the nuclei of pancreatic cancer cells. Weobserved that the nucleus-targeted, stimuli-responsive polymersomes released 70% ofencapsulated contents in the nucleus-mimicking environment in 80 min. We encapsulatedthe cancer stemness inhibitor BBI608 in the vesicles and observed that the BBI608encapsulated polymersomes reduced the viability of the BxPC3 cells to 43% in three-dimensional spheroid cultures. Thepolymersomes were prepared following a special protocol so that they scatter ultrasound, allowing imaging by a medicalultrasound scanner. Therefore, these echogenic, targeted, stimuli-responsive, and drug-encapsulated polymersomes have thepotential for trackable, targeted carrier of chemotherapeutic drugs to cancer cell nuclei.

Pullan J, Confeld M, Osborn J,  Kim J, Sarkar K,  Mallik S, 2019 “Exosomes as drug carriers for cancer therapy,” Molecular Pharamaceutics, 16, 1789-1798.

Exosomes, biological extracellular vesicles, have recently begun to find use in targeted drug delivery in solid tumor research. Ranging from 30−120 nm in size, exosomes are secreted from cells and
isolated from bodily fluids. Exosomes provide a unique material platform due to their characteristics, including physical properties such as stability, biocompatibility, permeability, low toxicity, and low immunogenicity—all critical to the success of any nanoparticle drug
delivery system. In addition to traditional chemotherapeutics, natural products and RNA have been encapsulated for the treatment of breast, pancreatic, lung, prostate cancers, and glioblastoma. This review discusses current research on exosomes for drug delivery to solid
tumors.

Pullan J, Confeld M, Osborn J,  Kim J, Sarkar K,  Mallik S, 2019 “Exosomes as drug carriers for cancer therapy,” Molecular Pharamaceutics, 16, 1789-1798.

Exosomes, biological extracellular vesicles, have recently begun to find use in targeted drug delivery in solid tumor research. Ranging from 30−120 nm in size, exosomes are secreted from cells and
isolated from bodily fluids. Exosomes provide a unique material platform due to their characteristics, including physical properties such as stability, biocompatibility, permeability, low toxicity, and low immunogenicity—all critical to the success of any nanoparticle drug
delivery system. In addition to traditional chemotherapeutics, natural products and RNA have been encapsulated for the treatment of breast, pancreatic, lung, prostate cancers, and glioblastoma. This review discusses current research on exosomes for drug delivery to solid
tumors.

Kulkarni P, Haldar MK, Karandish F, Confeld M, Hossain R, Borowicz P, Gange KN, Xia L, Sarkar K, Mallik S 2018 “Tissue-penetrating, hypoxia-responsive echogenic polymersomes for drug delivery to solid tumors,” Chemistry A European Journal, 24, 12490-12494.

Hypoxia in solid tumors facilitates the progres-sion 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 anti cancer drugs to solid tumors. The polymersomes are composed of a hy-poxia-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.

Karandish F, Haldar MK, Xia L, Gange KN, Feng L, You S, Choi Y, Sarkar K, Mallik S  2018 “Nucleus-targeted, echogenic polymersomes for delivering a cancer stemness inhibitor to pancreatic cancer cells,” Biomacromolecules, 19,4122-4132.

Chemotherapeutic agents for treating cancers show considerable sideeffects, toxicity, and drug resistance. To mitigate the problems, we designed nucleus-targeted, echogenic, stimuli-responsive polymeric vesicles (polymersomes) to transport andsubsequently release the encapsulated anticancer drugs within the nuclei of pancreaticcancer cells. We synthesized an alkyne-dexamethasone derivative and conjugated it to N3−polyethylene glycol (PEG)−polylactic acid (PLA) copolymer employing the Cu2+catalyzed“Click”reaction. We prepared polymersomes from the dexamethasone−PEG−PLA conjugate along with a synthesized stimuli-responsive polymer PEG−S−S−PLA. Thedexamethasone group dilates the nuclear pore complexes and transports the vesicles to thenuclei. We designed the polymersomes to release the encapsulated drugs in the presence ofa high concentration of reducing agents in the nuclei of pancreatic cancer cells. Weobserved that the nucleus-targeted, stimuli-responsive polymersomes released 70% ofencapsulated contents in the nucleus-mimicking environment in 80 min. We encapsulatedthe cancer stemness inhibitor BBI608 in the vesicles and observed that the BBI608encapsulated polymersomes reduced the viability of the BxPC3 cells to 43% in three-dimensional spheroid cultures. Thepolymersomes were prepared following a special protocol so that they scatter ultrasound, allowing imaging by a medicalultrasound scanner. Therefore, these echogenic, targeted, stimuli-responsive, and drug-encapsulated polymersomes have thepotential for trackable, targeted carrier of chemotherapeutic drugs to cancer cell nuclei.

Kumar KN, Mallik S, Sarkar K, 2017 “Role of freeze-drying in the presence of mannitol on the echogenicity of echogenic liposomes,” Journal of the Acoustical Society of America, 142, 3670-3676.

Echogenic liposomes (ELIPs) are an excellent candidate for ultrasound activated therapeutics andimaging. Although multiple experiments have established their echogenicity, the underlying mech-anism has remained unknown. However, freeze-drying in the presence of mannitol during ELIPpreparation has proved critical to ensuring echogenicity. Here, the role of this key component in thepreparation protocol was investigated by measuring scattering from freshly prepared freeze-driedaqueous solution of mannitol—and a number of other excipients commonly used in lyophiliza-tion—directly dispersed in water without any lipids in the experiment. Mannitol, meso-erythritol,glycine, and glucose that form a highly porous crystalline phase upon freeze-drying generated bub-bles resulting in strong echoes during their dissolution. On the other hand, sucrose, trehalose, andxylitol, which become glassy while freeze-dried, did not. Freeze-dried mannitol and other crystal-line substances, if thawed before being introduced into the scattering volume, did not produce echo-genicity, as they lost their crystallinity in the thawed state. The echogenicity disappeared in adegassed environment. Higher amounts of sugar in the original aqueous solution before freeze-drying resulted in higher echogenicity because of the stronger supersaturation and crystallinity. Thebubbles created by the freeze-dried mannitol in the ELIP formulation play a critical role in makingELIPs echogenic.

Xia L, Karandish F, Kumar KN, Froberg J, Kulkarni P, Gange KN, Choi Y, Mallik S, Sarkar K 2017 “Acoustic characterization of echogenic polymersomes prepared from amphiphilic block copolymers,” Ultrasound in Medicine and Biology, 44, 447-457.

Polymersomes are a class of artificial vesicles prepared from amphiphilic polymers. Like lipid vesicles(liposomes), they too can encapsulate hydrophilic and hydrophobic drug molecules in the aqueous core and thehydrophobic bilayer respectively, but are more stable than liposomes. Although echogenic liposomes have beenwidely investigated for simultaneous ultrasound imaging and controlled drug delivery, the potential of thepolymersomes remains unexplored. We prepared two different echogenic polymersomes from the amphiphilic co-polymers polyethylene glycol–poly-DL-lactic acid (PEG-PLA) and polyethylene glycol–poly-L-lactic acid (PEG-PLLA), incorporating multiple freeze-dry cycles in the synthesis protocol to ensure their echogenicity. We investigatedacoustic behavior with potential applications in biomedical imaging. We characterized the polymeric vesicles acous-tically with three different excitation frequencies of 2.25, 5 and 10 MHz at 500 kPa. The polymersomes exhibitedstrong echogenicity at all three excitation frequencies (about 50- and 25-dB enhancements in fundamental andsubharmonic, respectively, at 5-MHz excitation from 20g/mL polymers in solution). Unlike echogenic lipo-somes, they emitted strong subharmonic responses. The scattering results indicated their potential as contrastagents, which was also confirmed by clinical ultrasound imaging.

Nahire R, Hossain R, Patel R, Paul S, Ambre AH, Meghnani V, Layek B, Katti KS, Gange KN, Leclarc E, Srivastava D K, Sarkar K, Mallik S 2014 “Multifunctional polymersomes for cytosolic delivery of gemcitabine and doxorubicin to cancer cells,” Biomaterials, 35, 6482-6497.

Although liposomes are widely used as carriers of drugs and imaging agents, they suffer from a lack ofstability and the slow release of the encapsulated contents at the targeted site. Polymersomes (vesicles ofamphiphilic polymers) are considerably more stable compared to liposomes; however, they alsodemonstrate 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 torelease 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 amonolayer as well as in three-dimensional spheroid cultures. Polymersomes encapsulated with theanticancer drugs gemcitabine and doxorubicin showed significant cytotoxicity to these cells. Withfurther improvements, these vesicles hold the promise to serve as multifunctional nanocarriers, offeringa triggered release as well as diagnostic ultrasound imaging.

Nahire R, Halder M, Paul S, Margoum A, Ambre AH, Katti KS, Gange KN, Srivastava D K, Sarkar K, Mallik S 2014 “pH-Triggered Echogenicity and Contents Release from Liposomes,” Molecular Pharamaceutics, 11, 4059-4068.

Paul S, Nahire R, Mallik S, Sarkar K 2014 “Encapsulated microbubbles and echogenic liposomes for contrast ultrasound imaging and targeted drug delivery,” Computational Mechanics, 53,413-435.

Nahire R, Halder M, Paul S, Margoum A, Ambre AH, Katti KS, Gange KN, Srivastava D K, Sarkar K, Mallik S 2013 “Polymer Coated Echogenic Lipid Nanoparticles with dual release triggers,” Biomacromolecules, 14, 841-853.

Although lipid nanoparticles are promising drugdelivery vehicles, passive release of encapsulated contents atthe target site is often slow. Herein, we report contents releasefrom targeted, polymer-coated, echogenic lipid nanoparticles inthe cell cytoplasm by redox trigger and simultaneouslyenhanced by diagnostic frequency ultrasound. The lipidnanoparticles were polymerized on the external leaflet usinga disulfide cross-linker. In the presence of cytosolicconcentrations of glutathione, the lipid nanoparticles released76% of encapsulated contents. Plasma concentrations ofglutathione failed to releasethe encapsulated contents.Application of 3 MHz ultrasound for 2 min simultaneouslywith the reducing agent enhanced the release to 96%. Folicacid conjugated, doxorubicin-loaded nanoparticles showed enhanced uptake and higher cytotoxicity in cancer cells overexpressingthe folate receptor (compared to the control). With further developments, these lipid nanoparticles have the potential to be usedas multimodal nanocarriers for simultaneous targeted drug delivery and ultrasound imaging.

Chatterjee D, Sarkar K, Jain P 2005 “Ultrasound-mediated destruction of contrast microbubbles used for medical imaging and drug delivery,” Physics of Fluids, 17,100603.

Nahire R, Paul S, Scott M, Singh R, Muhonen W, Shabb J, Gange K, Srivastava D K, Sarkar K, Mallik S 2012 “Ultrasound enhanced matrix Metalloproteinase-9 Triggered Release of Contents from Echogenic Liposomes,” Molecular Pharmaceutics, 9, 2554-2564.

Nahire R, Halder M, Paul S, Margoum A, Ambre AH, Katti KS, Gange KN, Srivastava D K, Sarkar K, Mallik S 2013 “Polymer Coated Echogenic Lipid Nanoparticles with dual release triggers,” Biomacromolecules, 14, 841-853.

Although lipid nanoparticles are promising drugdelivery vehicles, passive release of encapsulated contents atthe target site is often slow. Herein, we report contents releasefrom targeted, polymer-coated, echogenic lipid nanoparticles inthe cell cytoplasm by redox trigger and simultaneouslyenhanced by diagnostic frequency ultrasound. The lipidnanoparticles were polymerized on the external leaflet usinga disulfide cross-linker. In the presence of cytosolicconcentrations of glutathione, the lipid nanoparticles released76% of encapsulated contents. Plasma concentrations ofglutathione failed to releasethe encapsulated contents.Application of 3 MHz ultrasound for 2 min simultaneouslywith the reducing agent enhanced the release to 96%. Folicacid conjugated, doxorubicin-loaded nanoparticles showed enhanced uptake and higher cytotoxicity in cancer cells overexpressingthe folate receptor (compared to the control). With further developments, these lipid nanoparticles have the potential to be usedas multimodal nanocarriers for simultaneous targeted drug delivery and ultrasound imaging.

Paul S, Nahire R, Mallik S, Sarkar K 2014 “Encapsulated microbubbles and echogenic liposomes for contrast ultrasound imaging and targeted drug delivery,” Computational Mechanics, 53,413-435.

Nahire R, Halder M, Paul S, Margoum A, Ambre AH, Katti KS, Gange KN, Srivastava D K, Sarkar K, Mallik S 2014 “pH-Triggered Echogenicity and Contents Release from Liposomes,” Molecular Pharamaceutics, 11, 4059-4068.

Nahire R, Hossain R, Patel R, Paul S, Ambre AH, Meghnani V, Layek B, Katti KS, Gange KN, Leclarc E, Srivastava D K, Sarkar K, Mallik S 2014 “Multifunctional polymersomes for cytosolic delivery of gemcitabine and doxorubicin to cancer cells,” Biomaterials, 35, 6482-6497.

Although liposomes are widely used as carriers of drugs and imaging agents, they suffer from a lack ofstability and the slow release of the encapsulated contents at the targeted site. Polymersomes (vesicles ofamphiphilic polymers) are considerably more stable compared to liposomes; however, they alsodemonstrate 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 torelease 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 amonolayer as well as in three-dimensional spheroid cultures. Polymersomes encapsulated with theanticancer drugs gemcitabine and doxorubicin showed significant cytotoxicity to these cells. Withfurther improvements, these vesicles hold the promise to serve as multifunctional nanocarriers, offeringa triggered release as well as diagnostic ultrasound imaging.

Xia L, Karandish F, Kumar KN, Froberg J, Kulkarni P, Gange KN, Choi Y, Mallik S, Sarkar K 2017 “Acoustic characterization of echogenic polymersomes prepared from amphiphilic block copolymers,” Ultrasound in Medicine and Biology, 44, 447-457.

Polymersomes are a class of artificial vesicles prepared from amphiphilic polymers. Like lipid vesicles(liposomes), they too can encapsulate hydrophilic and hydrophobic drug molecules in the aqueous core and thehydrophobic bilayer respectively, but are more stable than liposomes. Although echogenic liposomes have beenwidely investigated for simultaneous ultrasound imaging and controlled drug delivery, the potential of thepolymersomes remains unexplored. We prepared two different echogenic polymersomes from the amphiphilic co-polymers polyethylene glycol–poly-DL-lactic acid (PEG-PLA) and polyethylene glycol–poly-L-lactic acid (PEG-PLLA), incorporating multiple freeze-dry cycles in the synthesis protocol to ensure their echogenicity. We investigatedacoustic behavior with potential applications in biomedical imaging. We characterized the polymeric vesicles acous-tically with three different excitation frequencies of 2.25, 5 and 10 MHz at 500 kPa. The polymersomes exhibitedstrong echogenicity at all three excitation frequencies (about 50- and 25-dB enhancements in fundamental andsubharmonic, respectively, at 5-MHz excitation from 20g/mL polymers in solution). Unlike echogenic lipo-somes, they emitted strong subharmonic responses. The scattering results indicated their potential as contrastagents, which was also confirmed by clinical ultrasound imaging.

Kumar KN, Mallik S, Sarkar K, 2017 “Role of freeze-drying in the presence of mannitol on the echogenicity of echogenic liposomes,” Journal of the Acoustical Society of America, 142, 3670-3676.

Echogenic liposomes (ELIPs) are an excellent candidate for ultrasound activated therapeutics andimaging. Although multiple experiments have established their echogenicity, the underlying mech-anism has remained unknown. However, freeze-drying in the presence of mannitol during ELIPpreparation has proved critical to ensuring echogenicity. Here, the role of this key component in thepreparation protocol was investigated by measuring scattering from freshly prepared freeze-driedaqueous solution of mannitol—and a number of other excipients commonly used in lyophiliza-tion—directly dispersed in water without any lipids in the experiment. Mannitol, meso-erythritol,glycine, and glucose that form a highly porous crystalline phase upon freeze-drying generated bub-bles resulting in strong echoes during their dissolution. On the other hand, sucrose, trehalose, andxylitol, which become glassy while freeze-dried, did not. Freeze-dried mannitol and other crystal-line substances, if thawed before being introduced into the scattering volume, did not produce echo-genicity, as they lost their crystallinity in the thawed state. The echogenicity disappeared in adegassed environment. Higher amounts of sugar in the original aqueous solution before freeze-drying resulted in higher echogenicity because of the stronger supersaturation and crystallinity. Thebubbles created by the freeze-dried mannitol in the ELIP formulation play a critical role in makingELIPs echogenic.

Karandish F, Haldar MK, Xia L, Gange KN, Feng L, You S, Choi Y, Sarkar K, Mallik S  2018 “Nucleus-targeted, echogenic polymersomes for delivering a cancer stemness inhibitor to pancreatic cancer cells,” Biomacromolecules, 19,4122-4132.

Chemotherapeutic agents for treating cancers show considerable sideeffects, toxicity, and drug resistance. To mitigate the problems, we designed nucleus-targeted, echogenic, stimuli-responsive polymeric vesicles (polymersomes) to transport andsubsequently release the encapsulated anticancer drugs within the nuclei of pancreaticcancer cells. We synthesized an alkyne-dexamethasone derivative and conjugated it to N3−polyethylene glycol (PEG)−polylactic acid (PLA) copolymer employing the Cu2+catalyzed“Click”reaction. We prepared polymersomes from the dexamethasone−PEG−PLA conjugate along with a synthesized stimuli-responsive polymer PEG−S−S−PLA. Thedexamethasone group dilates the nuclear pore complexes and transports the vesicles to thenuclei. We designed the polymersomes to release the encapsulated drugs in the presence ofa high concentration of reducing agents in the nuclei of pancreatic cancer cells. Weobserved that the nucleus-targeted, stimuli-responsive polymersomes released 70% ofencapsulated contents in the nucleus-mimicking environment in 80 min. We encapsulatedthe cancer stemness inhibitor BBI608 in the vesicles and observed that the BBI608encapsulated polymersomes reduced the viability of the BxPC3 cells to 43% in three-dimensional spheroid cultures. Thepolymersomes were prepared following a special protocol so that they scatter ultrasound, allowing imaging by a medicalultrasound scanner. Therefore, these echogenic, targeted, stimuli-responsive, and drug-encapsulated polymersomes have thepotential for trackable, targeted carrier of chemotherapeutic drugs to cancer cell nuclei.

Kulkarni P, Haldar MK, Karandish F, Confeld M, Hossain R, Borowicz P, Gange KN, Xia L, Sarkar K, Mallik S 2018 “Tissue-penetrating, hypoxia-responsive echogenic polymersomes for drug delivery to solid tumors,” Chemistry A European Journal, 24, 12490-12494.

Hypoxia in solid tumors facilitates the progres-sion 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 anti cancer drugs to solid tumors. The polymersomes are composed of a hy-poxia-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.

Pullan J, Confeld M, Osborn J,  Kim J, Sarkar K,  Mallik S, 2019 “Exosomes as drug carriers for cancer therapy,” Molecular Pharamaceutics, 16, 1789-1798.

Exosomes, biological extracellular vesicles, have recently begun to find use in targeted drug delivery in solid tumor research. Ranging from 30−120 nm in size, exosomes are secreted from cells and
isolated from bodily fluids. Exosomes provide a unique material platform due to their characteristics, including physical properties such as stability, biocompatibility, permeability, low toxicity, and low immunogenicity—all critical to the success of any nanoparticle drug
delivery system. In addition to traditional chemotherapeutics, natural products and RNA have been encapsulated for the treatment of breast, pancreatic, lung, prostate cancers, and glioblastoma. This review discusses current research on exosomes for drug delivery to solid
tumors.

Pullan J, Dailey K, Bhallamudi S, Feng L, Alhalhooly L, Froberg J, Osborn J, Sarkar K, Molden T, Sathish V, Choi Y, Brooks A, Mallik S 2022, "Modified Bovine Milk Exosomes for Doxorubicin Delivery to Triple Negative Breast Cancer Cells" ACS Applied Bio Materials, 5, 2163-2175

Biological nanoparticles, such as exosomes, offer an approach to drug delivery because of their innate ability to transport biomolecules. Exosomes are derived from cells and an integral component of cellular communication. However, the cellular cargo of human exosomes could negatively impact their use as a safe drug carrier. Additionally, exosomes have the intrinsic yet enigmatic, targeting characteristics of complex cellular communication. Hence, harnessing the natural transport abilities of exosomes for drug delivery requires predictably targeting these biological nanoparticles. This manuscript describes the use of two chemical modifications, incorporating a neuropilin receptor agonist peptide (iRGD) and a hypoxiaresponsive lipid for targeting and release of an encapsulated drug from bovine milk exosomes to triple-negative breast cancer cells. Triple-negative breast cancer is a very aggressive and deadly form of malignancy with limited treatment options. Incorporation of both the iRGD peptide and hypoxiaresponsive lipid into the lipid bilayer of bovine milk exosomes and encapsulation of the anticancer drug, doxorubicin, created the peptide targeted, hypoxia-responsive bovine milk exosomes, iDHRX. Initial studies confirmed the presence of iRGD peptide and the exosomes’ ability to target the αvβ3 integrin, overexpressed on triple-negative breast cancer cells’ surface. These modified exosomes were stable under normoxic conditions but fragmented in the reducing microenvironment created by 10 mM glutathione. In vitro cellular internalization studies in monolayer and three-dimensional (3D) spheroids of triple-negative breast cancer cells confirmed the cell-killing ability of iDHRX. Cell viability of 50% was reached at 10 μM iDHRX in the 3D spheroid models using four different triple-negative breast cancer cell lines. Overall, the tumor penetrating, hypoxia-responsive exosomes encapsulating doxorubicin would be effective in reducing triple-negative breast cancer cells’ survival.

Pullan J, Dailey K, Bhallamudi S, Feng L, Alhalhooly L, Froberg J, Osborn J, Sarkar K, Molden T, Sathish V, Choi Y, Brooks A, Mallik S 2022, "Modified Bovine Milk Exosomes for Doxorubicin Delivery to Triple Negative Breast Cancer Cells" ACS Applied Bio Materials, 5, 2163-2175

Biological nanoparticles, such as exosomes, offer an approach to drug delivery because of their innate ability to transport biomolecules. Exosomes are derived from cells and an integral component of cellular communication. However, the cellular cargo of human exosomes could negatively impact their use as a safe drug carrier. Additionally, exosomes have the intrinsic yet enigmatic, targeting characteristics of complex cellular communication. Hence, harnessing the natural transport abilities of exosomes for drug delivery requires predictably targeting these biological nanoparticles. This manuscript describes the use of two chemical modifications, incorporating a neuropilin receptor agonist peptide (iRGD) and a hypoxiaresponsive lipid for targeting and release of an encapsulated drug from bovine milk exosomes to triple-negative breast cancer cells. Triple-negative breast cancer is a very aggressive and deadly form of malignancy with limited treatment options. Incorporation of both the iRGD peptide and hypoxiaresponsive lipid into the lipid bilayer of bovine milk exosomes and encapsulation of the anticancer drug, doxorubicin, created the peptide targeted, hypoxia-responsive bovine milk exosomes, iDHRX. Initial studies confirmed the presence of iRGD peptide and the exosomes’ ability to target the αvβ3 integrin, overexpressed on triple-negative breast cancer cells’ surface. These modified exosomes were stable under normoxic conditions but fragmented in the reducing microenvironment created by 10 mM glutathione. In vitro cellular internalization studies in monolayer and three-dimensional (3D) spheroids of triple-negative breast cancer cells confirmed the cell-killing ability of iDHRX. Cell viability of 50% was reached at 10 μM iDHRX in the 3D spheroid models using four different triple-negative breast cancer cell lines. Overall, the tumor penetrating, hypoxia-responsive exosomes encapsulating doxorubicin would be effective in reducing triple-negative breast cancer cells’ survival.

Sarkar K 2025, "Dynamics of bubbles and ultrasound: Diagnostic imaging to blood pressure monitoring and tissue engineering" Physical Review Fluids, 10, 030501

Coated microbubbles in conjunction with ultrasound have emerged as an important biomedical tool for diagnostic imaging and therapeutics. Here, I offer my perspective based on research spanning over two decades, highlighting the underlying physics of linear and nonlinear bubble dynamics. I will describe our effort at mathematical modeling of contrast microbubble behaviors, specifically our adoption of an interfacial rheological model for the stabilizing shell and a hierarchical approach of model building and improvement using attenuation and scattering experiments. I will describe our collaborative effort at using the sensitivity of the acoustic response of microbubbles to ambient hydrostatic pressure for a subharmonic aided pressure estimation (SHAPE) method for noninvasive organlevel blood pressure monitoring. Other collaborative projects demonstrated microbubbles together with low-intensity pulsed ultrasound (LIPUS) to be an effective tool in tissue engineering, growing bone and cartilage from human mesenchymal stem cells in a 3D printed tissue engineering scaffold. We have theoretically investigated various underlying mechanisms for such bioeffects of ultrasound-insonated microbubbles, specifically microstreaming at low acoustic excitations and cavitating jet formation at high excitations for shelled microbubbles. Finally, I briefly describe my computational research on viscous and viscoelastic emulsions and suspensions of drops, vesicles, and biological cells.