Since the first report on activatable CPPs (ACPPs) in 2004, various methods of activation have been developed. Here, we provide an overview of the different ACPP strategies known to date and summarize the benefits, drawbacks, and future directions.

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Hydrogels are water-saturated polymer networks and extensively used in drug delivery, tissue repair engineering, and cell cultures. For encapsulation of drugs or cells, the possibility to form hydrogels in situ is very much desired. This can be achieved in numerous ways, including use of bioorthogonal chemistry to create polymer networks. Here we report a set of bioorthogonally clickable polymers that was designed with the aim to find a combination that could rapidly encapsulate cells in a three-dimensional manner to improve the preparation of hydrogels as tissue mimics. To this end, tetrazine (Tet), trans-cyclooctene (TCO), azide (N3), dibenzocyclooctyne (DBCO), bicyclo[6.1.0]­nonyne (BCN), 3,4-dihydroxyphenylacetic acid (DHPA), and norbornene (Norb) were grafted to four-armed poly­(ethylene)­glycol (star-PEG) polymers of 10 kDa. Inverted vial tests and rheology demonstrated that hydrogels formed within seconds from combinations of TCO-Tet, BCN-DHPA, and BCN-Tet. Hydrogels from DBCO-N3, DBCO–DHPA, and BCN-N3 formed in the range of minutes, whereas the Norb-Tet ligation required multiple hours to form a gel. After this comparison, we chose to prepare hydrogels via DBCO-N3 and BCN-N3 and employed them for human mesenchymal stem cell (HMSC) cultures for a period of 5 days. We additionally incorporated RGDS and MMP cleavable peptide (MMPcp) motifs in these gels to stimulate cell adhesion and add degradability. Both DBCO and BCN gel systems including the functional peptide motifs allowed HMSCs to be viable and spread in 5 days. The DBCO-based hydrogel could trap cells at different depths due to its fast gelation process, while the slower gelation of the BCN-based hydrogel led to cell sedimentation.

The click reaction between a functionalized trans‐cyclooctene (TCO) and a tetrazine (Tz) is a compelling method for bioorthogonal conjugation in combination with payload releasing capabilities. However, the synthesis of difunctionalized TCOs remains challenging. As a result, these compounds are poorly accessible, which impedes the development of novel applications. In this work, the scalable and accessible synthesis of a new bioorthogonal difunctionalized TCO is reported in only four single selective high yielding steps starting from commercially available compounds. The TCO‐Tz click reaction was assessed and revealed excellent kinetic rates and subsequently payload release was shown with various functionalized derivatives. Tetrazine triggered release of carbonate and carbamate payloads was demonstrated up to 100 % release efficiency and local drug release was shown in a cellular toxicity study which revealed a >20‐fold increase in cytotoxicity.