Intracellular targeting of CD44+ cells with self-assembling, protein only nanoparticles

Pesarrodona, Mireia; Ferrer-Miralles, Neus; Unzueta, Ugutz; Gener, Petra; Tatkiewicz, Witold; Abasolo, Ibane; Ratera, Imma; Veciana, Jaume; Villaverde, Antonio; Vázquez, Esther

doi:10.1016/j.ijpharm.2014.07.016

Cited by 39 publications

(42 citation statements)

References 50 publications

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“…The de novo designed A5G27-GFP-H6 and T22-GFP-H6 nanoparticles show, by dynamic light scattering (DLS), average size peaks of 14 and 12 nm respectively when produced in the conventional E. coli strains BL21 (DE3) and Origami B respectively [19,20] (Supplementary Figure 1). Any intrinsic morphometric heterogeneity, if existing, has been so far unobserved and eclipsed in the analysis of the raw material.…”

Section: Resultsmentioning

confidence: 99%

“…The alternative morphometries in SEC peaks identified by SAXS were fully assessed by high resolution TEM and FESEM imaging (Figure 3), confirming the nano-architectonic variability of nanoparticles in a fully visual way. The protein nanoparticles studied here had been conceived as drug carriers for cancer treatments, what was lately encouraged by their good biodistribution when systemically administrated (upon which the material accumulated intracellularly in tumor and metastatic foci but not in liver, kidney, spleen or other nontarget organs) [19,20]. Then, how the morphometry and other physical properties of the oligomeric populations might influence receptor-dependent cell penetration is a critical issue that was addressed in a CXCR4 + cell culture model.…”

Section: Resultsmentioning

confidence: 99%

“…Protein production T22-GFP-H6 and A5G27-GFP-H6 are self-assembling modular proteins (Supplementary Figure 1) targeted to CXCR4 and CD44 respectively, through amino terminal peptides (T22, RRWCYRKCYKGYCYRKCR and A5G27, RLVSYNGIIFFLK) binding these cell surface receptors [20,21]. T22-GFP-H6 was produced in Escherichia coli Origami B [F − ompT hsdSB (rB − mB − ) gal dcm lacY1 ahpC (DE3) gor522::Tn10 trxB (Kan R , Tet R )] (Novagen, Madison, WI, USA), encoded in a pET22b-derived vector, and in the endotoxin-free KPM335 (msbA52, ΔgutQ, ΔkdsD, ΔlpxL, ΔlpxM, ΔpagP, ΔlpxP, ΔeptA, frr181), its parental BW30270 (CGSC#7925-MG1655; F − , rph + , fnr + ) and the routine wild type MC4100 (F − [araD139]B/r, Del(argF-lac)169, flhD5301, Δ(fruKyeiR)725(fruA25), relA1, rpsL150(str R ), rbsR22, Del(fimBfimE)632(::IS1), deoC1) from a pTrc99a-derivative plasmid.…”

Section: Methodsmentioning

confidence: 99%

“…Proteins were purified as previously described [20]. Briefly, cell pellets were resuspended in Wash Buffer (10 mM TrisHCl, 500 mM NaCl, 10 mM Imidazol pH 8.0 in presence of protease inhibitors.…”

Section: Protein Purificationmentioning

confidence: 99%

“…Such stability under physiological conditions, provided by complex forces sustaining protein-protein cross-interactions, enable the material escaping from renal clearance and it also allows prolonged circulation time in blood [19]. When displaying appropriate peptide ligands of cell surface cancer markers CXCR4 or CD44 (T22 and A5G27 respectively) they specifically accumulate in primary tumor and metastasis in colorectal and mammary cancer models respectively [20,21], being suited for antitumoral drug delivery. The same platform has been used to construct fluorescent nanoparticles that cross the blood-brain barrier and target the brain [18].…”

Section: Introductionmentioning

confidence: 99%

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Intrinsic functional and architectonic heterogeneity of tumor-targeted protein nanoparticles

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Self-assembling proteins are gaining attention as building blocks for application-tailored nanoscale materials. This is mostly due to the biocompatibility, biodegradability, and functional versatility of peptide chains. Such a potential for adaptability is particularly high in the case of recombinant proteins, which are produced in living cells and are suitable for genetic engineering. However, how the cell factory itself and the particular protein folding machinery influence the architecture and function of the final material is still poorly explored. In this study we have used diverse analytical approaches, including small-angle X-ray scattering (SAXS) and field emission scanning electron microscopy (FESEM) to determine the fine architecture and geometry of recombinant, tumor-targeted protein nanoparticles of interest as drug carriers, constructed on a GFP-based modular scheme. A set of related oligomers were produced in alternative Escherichia coli strains with variant protein folding networks. This resulted in highly regular populations of morphometric types, ranging from 2.4 to 28 nm and from spherical- to rod-shaped materials. These differential geometric species, whose relative proportions were determined by the features of the producing strain, were found associated with particular fluorescence emission, cell penetrability and receptor specificity profiles. Then, nanoparticles with optimal properties could be analytically identified and further isolated from producing cells for use. The cell’s protein folding machinery greatly modulates the final geometry reached by the constructs, which in turn defines the key parameters and biological performance of the material.Peer ReviewedPostprint (author's final draft

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Protein Purificationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Intrinsic functional and architectonic heterogeneity of tumor-targeted protein nanoparticles

et al. 2017

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Targeting Antitumoral Proteins to Breast Cancer by Local Administration of Functional Inclusion Bodies

Pesarrodona

Jauset²,

Díaz‐Riascos

et al. 2019

Advanced Science

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Two structurally and functionally unrelated proteins, namely Omomyc and p31, are engineered as CD44‐targeted inclusion bodies produced in recombinant bacteria. In this unusual particulate form, both types of protein materials selectively penetrate and kill CD44+ tumor cells in culture, and upon local administration, promote destruction of tumoral tissue in orthotropic mouse models of human breast cancer. These findings support the concept of bacterial inclusion bodies as versatile protein materials suitable for application in chronic diseases that, like cancer, can benefit from a local slow release of therapeutic proteins.

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Endosomal escape of protein nanoparticles engineered through humanized histidine-rich peptides

et al. 2019

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Poly-histidine peptides such as H6 (HHHHHH) are used in protein biotechnologies as purification tags, protein-assembling agents and endosomal-escape entities. The pleiotropic properties of such peptides make them appealing to design protein-based smart materials or nanoparticles for imaging or drug delivery to be produced in form of recombinant proteins. However, the clinical applicability of H6tagged proteins is restricted by the potential immunogenicity of these segments. In this study, we have explored several humanized histidine-rich peptides in tumor-targeted modular proteins, which can specifically bind and be internalized by the target cells through the tumoral marker CXCR4. We were particularly interested in exploring how protein purification, self-assembling and endosomal escape perform in proteins containing the variant histidine-rich tags. Among the tested candidates, the peptide H5E (HEHEHEHEH) is promising as a good promoter of endosomal escape of the associated fulllength protein upon endosomal internalization. The numerical modelling of cell penetration and endosomal escape of the tested proteins has revealed a negative relationship between the amount of protein internalized into target cells and the efficiency of cytoplasmic release. This fact demonstrates that the His-mediated, proton sponge-based endosomal escape saturates at moderate amounts of internalized protein, a fact that might be critical for the design of protein materials for cytosolic molecular delivery.

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Intracellular targeting of CD44+ cells with self-assembling, protein only nanoparticles

Cited by 39 publications

References 50 publications

Intrinsic functional and architectonic heterogeneity of tumor-targeted protein nanoparticles

Intrinsic functional and architectonic heterogeneity of tumor-targeted protein nanoparticles

Targeting Antitumoral Proteins to Breast Cancer by Local Administration of Functional Inclusion Bodies

Endosomal escape of protein nanoparticles engineered through humanized histidine-rich peptides

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