An Oligonucleotide Transfection Vector Based on HSA and PDMAEMA Conjugation: Effect of Polymer Molecular Weight on Cell Proliferation and on Multicellular Tumor Spheroids

Abstract

A novel gene transfection vector was fabricated based on the conjugation of human serum albumin (HSA) and maleimide end functionalized poly[(N,N-dimethylamino) ethyl methacrylate] (PDMAEMA). The bioconjugation was achieved in a site-specific manner to yield well-defined polymer-protein conjugates. The biohybrid was able to bind DNA with high affinity resulting in nanoparticles with a HSA shell. This paper mainly focuses on the influence of polymeric chain length on the particle properties and their drug-carrying ability to deliver oligonucleotides into breast cancer cells. The cytotoxic agent of interest, ISIS5132, is an oligonucleotide which disrupts DNA function within the cell. There was no evidence that the polymeric chain length had any effects on the conjugation efficiency and the subsequent condensation ability of the conjugates to oligonucleotide. However, the polymeric chain length had an obvious effect on the size of the complex micelles. Low molecular weights only led to loosely compacted complexes with the oligonucleotide, while large molecular weight led to well-defined nanoparticle structures. More importantly, it was found that the variation in the length of the PDMAEMA block resulted in a change in cytotoxicity of the drug loaded complex micelle. That is, the concentration of 50% inhibition (IC50 ) of the complex micelle on MDA-MB-231 and MCF-7 cells reached the lowest value at a chain length of around 21 000 g mol(-1) . The IC50 value increased when the polymeric chain length was shorter (8000 g mol(-1) and 10 000 g mol(-1) ) while it increased again when PDMAMEA of M¯n = 47 000 g mol(-1) , probably due to insufficient release of the drug. These result were reflected when investigating the performance of the polyplex using MCF-7 multicellular tumor spheroids, where again the medium PDMAEMA chain length led to the best delivery vehicle for ISIS5132.

Multicellular tumor spheroids: a relevant 3D model for the in vitro preclinical investigation of polymer nanomedicines

“…44,50 Therefore, once formed, spheroids need to be transferred into 96-well plates or embedded in polymer matrices for further studies. [88][89][90][91][92][93][94][95] Not only such transfer can affect the integrity of spheroids but it clearly makes this technique extremely labor intensive and time-consuming, thus preventing large scale application. 44,50,51 Commercially available platforms (InSphero GravityPlus™ technology and PERFECTA3D™ hanging drop plates of 3D Biomatrix) can reduce the workload and enable experiments to be carried out in a high-throughput manner, although with a significant increase of the expenses.…”

“…37,[39][40][41]45 Optical microscopy is the simplest and most widely used technique for the visual assessment of morphological changes in spheroids. 52,[55][56][57][58][60][61][62][63][64][65]70,73,74,77,84,[89][90][91][92][93]95,107,110,[114][115][116][117][118][119][120][121][122][123][124][125][126] Following exposure to various treatments, the measurement of the variation of morphometric parameters (e.g., mean diameter, minimum diameter, maximum diameter, volume, area and circularity) of spheroids from bright field images is used for a quantitative analysis of the extent of growth inhibition. 37,87 After interruption of the treatment, the spheroid growth delay might be calculated in comparison with untreated samples as the time lag required to reach the quintuple spheroid volume of day 4 (5 × V d4 ; assuming V d4 as the starting spheroid volume at the onset of treatment).…”

“…Complexation has been realized for instance with (i) poly(amidoamine) (PAMAM) dendrimers; 92,149 (ii) folate-functionalized-poly(ethylene glycol)-polyamidoamine-polyethylenimine (PME-(PEG-FA)) copolymers 94 (see also section 2.3.6.2); (iii) poly(L-lysine); 118 (iv) PEG-b-poly(Nsubstituted asparagine) copolymers(PEG-b-P[Asp(DET)); 131 (v) triblock poly(2-ethyl-2-oxazoline)-poly(L-lactide)-g-poly(ethylenimine) (PEOz-PLA-g-PEI) polymers; 108,109 (vi) mPEG-PEI (CendRpenetrating-peptide-modified methoxy poly(ethylene glycol)polyethylenimine) copolymers 80 (see also section 2.3.6.6); (vii) folate-poly(ethylene glycol) (PEG)-Amino Acid Modified Chitosan (CM-PFA) 107 or (viii) poly[(N,N-dimethylamino) ethyl methacrylate] (PDMAEMA)-derivatized albumin. 93,116 The pioneering studies of Kataoka and coworkers have highlighted the utility of human hepatocarcinoma multicellular spheroids (HuH-7 cells) for the long-term evaluation of the transfection efficacy achieved using polyplex micelles as gene delivery systems. 118,131 In particular, core-shell type micelles assembled through electrostatic interactions between poly (L-lysine) and lactosylated poly(ethylene glycol)-siRNA conjugate (lac-PEGylated polyplexes) 118 or pDNA and PEG-block-poly (N-asparagine) copolymers (PEG-b-P[Asp(DET)]) 131 have been investigated.…”

Drug Delivery Vehicles Based on Albumin–Polymer Conjugates

“…Poly(2‐dimethylaminoethyl methacrylate) (PDMAEMA) with tunable polymeric chain length was conjugated to HSA or BSA via a maleimide endfunctionality and subsequently complexed with oligonucleotide ISIS 5132. [31a], The polymer chain length was found to be a critical parameter influencing the particle size, stability, drug loading, and releasing efficiency and subsequent cytotoxic behavior of the self‐assembled albumin–polymer nanoparticles. Bioconjugates with longer PDMAEMA chain length resulted in larger complex micelles with high stability and a narrower size distribution compared to bioconjugates with shorter chain length polymers.…”

Lipid–Albumin Nanoparticles (LAN) for Therapeutic Delivery of Antisense Oligonucleotide against HIF-1α

“…Recently, albumin has been used for the delivery of ASOs and siRNAs. − It is considered to be an ideal material for in vivo applications, because albumin is naturally abundant in serum. Albumin also possesses endosomal lytic activity due to its hydrophobic core, which upon conformational change can be exposed and can induce bilayer disruption or membrane fusion.…”