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PROTEIN-NANOPARTICLE CO-ENGINEERING: SELF-ASSEMBLY, INTRACELLULAR PROTEIN DELIVERY, AND CRISPR/CAS9-BASED GENE EDITING
Direct cytoplasmic delivery of gene editing nucleases such CRISPR/Cas9 systems and therapeutic proteins provides enormous opportunities in curing human genetic diseases, and assist research in basic cell biology. One approach to attain such a goal is through engineering nanotechnological tools to mimic naturally existing intra- and extracellular protein delivery/transport systems. Nature builds transport systems for proteins and other biomolecules through evolution-derived sophisticated molecular engineering. Inspired by such natural assemblies, I employed molecular engineering approaches to fabricate self-assembled nanostructures to use as intracellular protein delivery tools. Briefly, proteins and gold nanoparticles were co-engineered to carry complementary electrostatic recognition elements. When these materials were mixed together, they formed highly sophisticated, multi-layered, and hierarchical self-assembled nanostructures of few hundred-nanometer size. These structures carried a large number of engineered proteins, got fused to cell membrane upon incubation, and delivered the encapsulated protein content directly into cell cytoplasm. Using this technology, we delivered a wide range of proteins, and CRISPR/Cas9-ribonucleoprotein that resulted high efficient gene editing
Anti-malarial activity of geldanamycin derivatives in mice infected with Plasmodium yoelii
Background
Geldanamycin (GA), a benzoquinone ansamycin antibiotic has been shown in vitro to possess anti-plasmodial activity. Pharmacological activity of this drug is attributed to its ability to inhibit PfHSP90. The parasite growth arrest has been shown to be due to drug-induced blockage of the transition from ring to trophozoite stage. To further evaluate the consequences of this pharmacodyamic feature, the anti-malarial activity of GA analogs with enhanced drug properties in a Plasmodium-infected animal model have been evaluated for their capacity to induce clearance of the parasite. In the process, a hypothesis was subsequently tested regarding the susceptibility of the cured animals to malaria reflected in an attenuated parasite load that may be evoked by a protective immune response in the host. Methods
Six weeks old Swiss mice were infected with a lethal Plasmodium yoelii (17XL) strain. On appearance of clinical symptoms of malaria, these animals were treated with two different GA derivatives and the parasite load was monitored over 15-16 days. Drug-treated animals cured of the parasite were then re-challenged with a lethal dose of P. yoelii 17XL. Serum samples from GA cured mice that were re-challenged with P. yoelii 17XL were examined for the presence of antibodies against the parasite proteins using western blot analysis. Results
Treatment of P. yoelii 17XL infected mice with GA derivatives showed slow recovery from clinical symptoms of the disease. Blood smears from drug treated mice indicated a dominance of ring stage parasites when compared to controls. Although, P. yoelii preferentially invades normocytes (mature rbcs), in drug-treated animals there was an increased invasion of reticulocytes. Cured animals exhibited robust protection against subsequent infection and serum samples from these animals showed antibodies against a vast majority of parasite proteins. Conclusions
Treatment with GA derivatives blocked the transition from ring to trophozoite stage presumably by the inhibition of HSP90 associated functions. Persistence of parasite in ring stage leads to robust humoral immune response as well as a shift in invasion specificity from normocytes to reticulocyte. It is likely that the treatment with the water-soluble GA derivative creates an attenuated state (less virulent with altered invasion specificity) that persists in the host system, allowing it to mount a robust immune response
Probing the proteinânanoparticle interface: the role of aromatic substitution pattern on affinity
General Strategy for Direct Cytosolic Protein Delivery <i>via</i> ProteinâNanoparticle Coâengineering
Endosomal entrapment is a key hurdle
for most intracellular protein-based
therapeutic strategies. We report a general strategy for efficient
delivery of proteins to the cytosol through co-engineering of proteins
and nanoparticle vehicles. The proteins feature an oligoÂ(glutamate)
sequence (E-tag) that binds arginine-functionalized gold nanoparticles,
generating hierarchical spherical nanoassemblies. These assemblies
fuse with cell membranes, releasing the E-tagged protein directly
into the cytosol. Five different proteins with diverse charges, sizes,
and functions were effectively delivered into cells, demonstrating
the generality of our method. Significantly, the engineered proteins
retained activity after cytosolic delivery, as demonstrated through
the delivery of active Cre recombinase, and granzyme A to kill cancer
cells
Rapid phenotyping of cancer stem cells using multichannel nanosensor arrays
Cancer stem cells (CSCs) contribute to multidrug resistance, tumor recurrence and metastasis, making them prime therapeutic targets. Their ability to differentiate and lose stem cell properties makes them challenging to study. Currently, there is no simple assay that can capture and trace the dynamic phenotypic changes on the CSC surface. Here, we report rapid discrimination of breast CSCs from non-CSCs using a nanoparticle-fluorescent-protein based sensor. This nanosensor was employed to discriminate CSCs from non-CSCs, as well CSCs that had differentiated in vitro in two breast cancer models. Importantly, the sensor platform could also discriminate CSCs from the bulk population of cells in patient-derived xenografts of human breast cancer. Taken together, the results obtained demonstrate the feasibility of using the nanosensor to phenotype CSCs and monitor their fate. Furthermore, this approach provides a novel area for therapeutic interventions against these challenging targets
Programmed Self-Assembly of Hierarchical Nanostructures through ProteinâNanoparticle Coengineering
Hierarchical organization
of macromolecules through self-assembly
is a prominent feature in biological systems. Synthetic fabrication
of such structures provides materials with emergent functions. Here,
we report the fabrication of self-assembled superstructures through
coengineering of recombinant proteins and nanoparticles. These structures
feature a highly sophisticated level of multilayered hierarchical
organization of the components: individual proteins and nanoparticles
coassemble to form discrete assemblies that collapse to form granules,
which then further self-organize to generate superstructures with
sizes of hundreds of nanometers. The components within these superstructures
are dynamic and spatially reorganize in response to environmental
influences. The precise control over the molecular organization of
building blocks imparted by this proteinânanoparticle coengineering
strategy provides a method for creating hierarchical hybrid materials
Probing the proteinânanoparticle interface: the role of aromatic substitution pattern on affinity
<div><p>A new class of cationic gold nanoparticles (NPs) has been synthesised bearing benzyl moieties featuring âNO<sub>2</sub> and âOMe groups to investigate the regioisomeric control of aromatic NPâprotein recognition. In general, NPs bearing electron-withdrawing groups demonstrated higher binding affinities towards green fluorescent protein (GFP) than NPs bearing electron-donating groups. Significantly, a âź7.5- and âź4.3-fold increase in binding with GFP was observed for âNO<sub>2</sub> groups in <i>meta-</i>position and <i>para-</i>position, respectively, while <i>ortho</i>-substitution showed binding similar to the unsubstituted ring. These findings demonstrated that the NPâprotein interaction can be controlled by tuning the spatial orientation and the relative electronic properties of the aromatic substituents. This improved biomolecular recognition provides opportunities for enhanced biosensing and functional protein delivery to the cells.</p></div
CRISPRed Macrophages for Cell-Based Cancer Immunotherapy
We present here an
integrated nanotechnology/biology strategy for
cancer immunotherapy that uses arginine nanoparticles (ArgNPs) to
deliver CRISPR-Cas9 gene editing machinery into cells to generate
SIRP-Îą knockout macrophages. The NP system efficiently codelivers
single guide RNA (sgRNA) and Cas9 protein required for editing to
knock out the âdonât eat me signalâ in macrophages
that prevents phagocytosis of cancer cells. Turning off this signal
increased the innate phagocytic capabilities of the macrophages by
4-fold. This improved attack and elimination of cancer cells makes
this strategy promising for the creation of âweaponizedâ
macrophages for cancer immunotherapy
Programmed Self-Assembly of Hierarchical Nanostructures through ProteinâNanoparticle Coengineering
Hierarchical organization
of macromolecules through self-assembly
is a prominent feature in biological systems. Synthetic fabrication
of such structures provides materials with emergent functions. Here,
we report the fabrication of self-assembled superstructures through
coengineering of recombinant proteins and nanoparticles. These structures
feature a highly sophisticated level of multilayered hierarchical
organization of the components: individual proteins and nanoparticles
coassemble to form discrete assemblies that collapse to form granules,
which then further self-organize to generate superstructures with
sizes of hundreds of nanometers. The components within these superstructures
are dynamic and spatially reorganize in response to environmental
influences. The precise control over the molecular organization of
building blocks imparted by this proteinânanoparticle coengineering
strategy provides a method for creating hierarchical hybrid materials
Direct Cytosolic Delivery of CRISPR/Cas9-Ribonucleoprotein for Efficient Gene Editing
Genome editing through
the delivery of CRISPR/Cas9-ribonucleoprotein
(Cas9-RNP) reduces unwanted gene targeting and avoids integrational
mutagenesis that can occur through gene delivery strategies. Direct
and efficient delivery of Cas9-RNP into the cytosol followed by translocation
to the nucleus remains a challenge. Here, we report a remarkably highly
efficient (âź90%) direct cytoplasmic/nuclear delivery of Cas9
protein complexed with a guide RNA (sgRNA) through the coengineering
of Cas9 protein and carrier nanoparticles. This construct provides
effective (âź30%) gene editing efficiency and opens up opportunities
in studying genome dynamics