Reverse-QTY code design of active human serum albumin self-assembled amphiphilic nanoparticles for effective anti-tumor drug doxorubicin release in mice

Human serum albumin (HSA) is a highly water-soluble protein with 67% alpha-helix content and three distinct domains (I, II, and III). HSA offers a great promise in drug delivery with enhanced permeability and retention effect. But it is hindered by protein denaturation during drug entrapment or conjugation that result in distinct cellular transport pathways and reduction of biological activities. Here we report using a protein design approach named reverse -QTY (rQTY) code to convert specific hydrophilic alpha-helices to hydrophobic to alpha-helices. The designed HSA undergo self-assembly of well-ordered nanoparticles with highly biological actives. The hydrophilic amino acids, asparagine (N), glutamine (Q), threonine (T), and tyrosine (Y) in the helical B-subdomains of HSA were systematically replaced by hydrophobic leucine (L), valine (V), and phenylalanine (F). HSArQTY nanoparticles exhibited efficient cellular internalization through the cell membrane albumin binding protein GP60, or SPARC (secreted protein, acidic and rich in cysteine)-mediated pathways. The designed HSArQTY variants displayed superior biological activities including: i) encapsulation of drug doxorubicin, ii) receptor-mediated cellular transport, iii) tumor cell targeting, and iv) antitumor efficiency compare to denatured HSA nanoparticles. HSArQTY nanoparticles provided superior tumor targeting and antitumor therapeutic effects compared to the albumin nanoparticles fabricated by antisolvent precipitation method. We believe that the rQTY code is a robust platform for specific hydrophobic modification of functional hydrophilic proteins with clear-defined binding interfaces.

Automated summary

In this study, a protein design approach called reverse-QTY (rQTY) code was used to convert hydrophilic alpha-helices in human serum albumin (HSA) to hydrophobic alpha-helices, resulting in the formation of well-ordered nanoparticles with high biological activity. The designed HSArQTY variants exhibited superior biological activities, including drug encapsulation, receptor-mediated cellular transport, tumor cell targeting, and antitumor efficiency, compared to denatured HSA nanoparticles. HSArQTY nanoparticles provided better tumor targeting and antitumor therapeutic effects compared to albumin nanoparticles fabricated by antisolvent precipitation method. The rQTY code is considered as a robust platform for specific hydrophobic modification of functional hydrophilic proteins with clear-defined binding interfaces.