Local Release of Highly Loaded Antibodies from Functionalized Nanoporous Support for Cancer Immunotherapy

Abstract

Recent advances with functionalized nanoporous supports provide an innovative approach for entrapping proteins and for their subsequent controlled release and delivery.1 -7 Functionalized mesoporous silica (FMS) can provide a confined and interactive nanoenvironment that increases protein activity and allow large amounts of protein loading compared to unfunctionalized mesoporous silica (UMS) or normal porous silica.5 -7 First, the proteins can be spontaneously entrapped in FMS with rigid, uniform, open nanopore geometry of tens of nanometers via non-covalent interaction. Then, one can control the release of the entrapped proteins from FMS based on the function groups and pore sizes when the FMS-protein composites are dispersed in a fresh buffer solution in which a new thermodynamic balance can be reached. In this work, we found that antibodies can be spontaneously loaded in FMS with super-high density (0.4-0.8 mg of antibody/mg of FMS) due to their comprehensive noncovalent interaction. We hypothesize that therapeutic antibodies entrapped in FMS can be gradually released locally in vivo under physiological conditions and that this will help develop innovative therapies for many diseases. We performed pilot tests to investigate the anti-tumor activity of a monoclonal antibody (mAb) to CTLA4,8 an immunoregulatory molecule released from FMS at the tumor site. This strategy resulted in much greater and extended inhibition of tumor growth than the antibody given systematically.To ensure large loading of mAb molecules (M.W. 150kDa) in FMS, we prepared UMS with a pore size (diameter) as large as 30 nm, a surface area as great as 533 m 2 /g and an average bead size of 12-15 μm (Supporting information).9 , 10 A controlled hydration and condensation reaction was used to introduce functional groups into UMS.9 ,10 Coverage of 2% (or 20%) HOOC-FMS, HO 3 S-FMS or NH 2 -FMS means 2% (or 20%) of the total available silanol groups (5 × 10 18 silanol groups per square meter9 , 10) of UMS would be silanized with trimethoxysilane with the functional group HOOC, HO 3 S or NH 2 . [1][2][3][4][5][6][7] Figs. 1A shows the TEM image of 30 nm 20% HOOC-FMS. There is no significant difference between the TEM images of UMS and their corresponding FMS. 6 Unlike 3-nm and 10-nm mesoporous silica, the 30-nm mesoporous silica has a large degree of disordering, 11 but it still reveals more or less uniform cage-like porous structure. 12 FMS was incubated in the antibody solution, where the antibody would be spontaneously entrapped in FMS. We defined the protein amount (mg) of an antibody entrapped with 1 mg * To whom correspondence should be addressed. chenghong.lei@pnl.gov; hellsk@u.washington.edu; jun.liu@pnl.gov.. 3 Current address: Karolinska Institutet, Department of Oncology-Pathology, SE171-76 Stockholm, Sweden. † These authors contribute equally to this work. Supporting Information Available:Experimental section and additional experimental data are available free of charge via the Internet at http://pubs.acs.org. of FMS as the protein-load...

A Vesicle Supra‐Assembly Approach to Synthesize Amine‐Functionalized Hollow Dendritic Mesoporous Silica Nanospheres for Protein Delivery

“…Driven by the electrostatic attraction between positively charged A‐HDMSN and negatively charged protein molecules, A‐HDMSN demonstrate a significantly higher loading capacity of 391 ± 16 μg mg −1 toward β‐Gal and 658 ± 45 μg mg −1 toward IgG ( Figure a), compared to C‐HDMSN (162 ± 19 μg mg −1 to β‐Gal and 416 ± 53 μg mg −1 to IgG). Those values are the highest among the currently reported results . The TGA analysis data (Figure S7, Supporting Information) show a weight loss of 60% contributed by immobilized IgG in A‐HDMSN, which is consistent with the value determined by adsorption method in Figure a.…”

Core‐Cone Structured Monodispersed Mesoporous Silica Nanoparticles with Ultra‐large Cavity for Protein Delivery

“…The IgG loading capacity of MSN-CC is comparable to that of silica foams with a large pore of 30 nm (≈610 mg g −1 ). [ 8 ] Besides high loading capacity, effi cient cellular uptake is another prerequisite factor for protein delivery. To investigate cellular internalization of the particles, MSN-CC were modifi ed with 3-aminopropyltriethoxysilane then conjugated with a red fl orescence molecule, rhodamine B isothicyanate (RITC).…”

“…Traditional MSNs usually have small pore sizes, thus their applications are limited for the delivery of small proteins . Foam‐like silica materials with a pore size of 30 nm have been utilized to load large proteins (Immunoglobulin G, IgG); however, their micrometer particle size is not desired for cellular delivery. Bale et al reported the successfully intracellular delivery of antibody into cells by hydrophobically functionalized solid silica nanoparticles; however the loading capacity was relatively low.…”

Curing Mice with Large Tumors by Locally Delivering Combinations of Immunomodulatory Antibodies

“…over systemic injection of immunomodulatory mAbs has also been demonstrated by others, primarily as an approach to reduce systemic toxicity (20, 45, 46). To further improve its therapeutic window one may obtain a sustained high intratumoral level by entrapping the mAbs in nanoparticles (47) or by using constructs that combine immunomodulatory mAbs with tumor-targeting ones; if the targeting is efficient, the constructs may be administered systemically.…”